Biocompatible poly-B-1 4-N-acetylglucosamine

ABSTRACT

The present invention relates to a purified, easily produced poly-β-1→4-N-acetylglucosamine (p-GlcNAc) polysaccharide species. The p-GlcNAc of the invention is a polymer of high molecular weight whose constituent monosaccharide sugars are attached in a β-1→4 conformation, and which is free of proteins, and substantially free of single amino acids, and other organic and inorganic contaminants. In addition, derivatives and reformulations of p-GlcNAc are described. The present invention further relates to methods for the purification of the p-GlcNAc of the invention from microalgae, preferably diatom, starting sources. Still further, the invention relates to methods for the derivatization and reformulation of the p-GlcNAc. Additionally, the present invention relates to the uses of pure p-GlcNAc, its derivatives, and/or its reformulations.

1. INTRODUCTION

[0001] The present invention relates, first, to a purified, easilyproduced poly-β-1→4-N-acetylglucosamine (p-GlcNAc) polysaccharidespecies. The p-GlcNAc of the invention is a polymer of high molecularweight whose constituent monosaccharide sugars are attached in a β-1→4conformation, and which is free of proteins, and substantially free ofsingle amino acids, and other organic and inorganic contaminants. Inaddition, derivatives and reformulations of p-GlcNAc are described. Thepresent invention further relates to methods for the purification of thep-GlcNAc of the invention from microalgae, preferably diatom, startingsources. Still further, the invention relates to methods for thederivatization and reformulation of the p-GlcNAc. Additionally, thepresent invention relates to the uses of pure p-GlcNAc, its derivatives,and/or its reformulations.

2. BACKGROUND OF THE INVENTION

[0002] There exists today an extensive literature on the properties,activities, and uses of polysaccharides that consist, in part, ofp-GlcNAc. A class of such materials has been generically referred to as“chitin”, while deacetylated chitin derivatives have been referred to as“chitosan”. When these terms were first used, around 1823, it wasbelieved that chitin and chitosan always occurred in nature as distinct,well-defined, unique, and invariant chemical species, with chitin beingfully acetylated and chitosan being fully deacetylated compositions. Itwas approximately a century later, however, before it was discoveredthat the terms “chitin” and “chitosan” are, in fact, very ambiguous.Rather than referring to well-defined compounds, these terms actuallyrefer to a family of compounds that exhibit widely differing physicaland chemical properties. These differences are due to the products'varying molecular weights, varying degrees of acetylation, and thepresence of contaminants such as covalently bound, species-specificproteins, single amino acid and inorganic contaminants. Even today, theterms “chitin” and “chitosan” are used ambiguously, and actually referto poorly defined mixtures of many different compounds.

[0003] For example, the properties of “chitins” isolated fromconventional sources such as crustacean outer shells and fungal mycelialmats are unpredictably variable. Such variations are due not only tospecies differences but are also due to varying environmental andseasonal effects that determine some of the biochemical characteristicsof the “chitin”-producing species. In fact, the unpredictablevariability of raw material is largely responsible for the slow growthof chitin-based industries.

[0004] No reports exist today in the scientific literature describingthe isolation and production, from material sources, of pure, fullyacetylated p-GlcNAc, i.e., a product or products uncontaminated byorganic or inorganic impurities. While McLachlan et al. (McLachlan, A.G. et al., 1965, Can. J. Botany 43:707-713) reported the isolation ofchitin, subsequent studies have shown that the “pure” substanceobtained, in fact contained proteins and other contaminants.

[0005] Deacetylated and partially deacetylated chitin preparationsexhibit potentially beneficial chemical properties, such as highreactivity, dense cationic charges, powerful metal chelating capacity,the ability to covalently attach proteins, and solubility in manyaqueous solvents. The unpredictable variability of these preparations,as described above, however, severely limits the utility of theseheterogenous compounds. For example, the currently available “chitins”and “chitosans” give rise to irreproducible data and to unacceptablywide variations in experimental results. Additionally, the availablepreparations are not sufficiently homogenous or pure, and thepreparation constituents are not sufficiently reproducible for thesepreparations to be acceptable for use in applications, especially inmedical ones. Thus, although extremely desirable, true, purifiedpreparations of chitin and chitosan, whose properties are highlyreproducible and which are easily manufactured, do not currently exist.

3. SUMMARY OF THE INVENTION

[0006] The present invention relates, first, to an isolated, easilyproduced, pure p-GlcNAc species. The p-GlcNAc of the invention is apolymer of high molecular weight whose constituent monosaccharides areattached in a β-1→4 conformation, and which is free of proteins,substantially free of other organic contaminants, and substantially freeof inorganic contaminants.

[0007] The importance of the present invention resides in the fact thatthe problem of unpredictable raw material variability has been overcome.It is, for the first time, possible to produce, by simple means, and ona commercial scale, biomedically pure, p-GlcNAc of high molecular weightand consistent properties. The material produced in the presentinvention is highly crystalline and is produced from carefullycontrolled, aseptic cultures of one of a number of marine microalgae,preferably diatoms, which have been grown in a defined medium.

[0008] The present invention further describes derivatives andreformulations of p-GlcNAc as well as methods for the production of suchderivatives and reformulations. Such derivatizations may include, butare not limited to polyglucosamine and its derivatives, and suchreformulations may include, but are not limited to membranes, filaments,non-woven textiles, sponges, gels and three-dimensional matrices. Stillfurther, the present invention relates to methods for the purificationof the p-GlcNAc of the invention from microalgae, preferably diatom,sources.

[0009] Additionally, the present invention relates to the uses of thepurified p-GlcNAc, its derivatives, and/or its reformulations. Amongthese uses are novel commercial applications relating to such industriesas the biomedical, pharmaceutical, cosmetic and agricultural industries,all of which require starting materials of the highest degree of purity.For example, the p-GlcNAc materials of the invention may be, formulatedto exhibit controllable biodegradation properties, and, further, may beused as part of slow drug delivery systems, as cell encapsulationsystems, and as treatments for the prevention of post-surgicaladhesions; and for the induction of hemostasis. For example, thep-GlcNAc materials of the invention exhibit properties that make themideally suited for a large number of biomedical applications. Some ofthese properties include but are not limited to: high purity andcomposition consistency; biocompatibility; controllablebiodegradability; and, an ability to immobilize and encapsulate agents,such as therapeutic agents, and cells. These properties are useful, forexample, in the formulation of biodegradable barrier devices, improveddrug formulations and cell based therapeutics.

[0010] The biodegradable barriers of the invention include p-GlcNAcbased materials used as devices, for example, as temporary barrierswhich become resorbed by the body. This category of products include,but is not limited to, devices that prevent the formation of surgicaladhesions, stop bleeding, and promote wound healing. Such biodegradablebarriers can further be used as surgical space fillers, peridontalbarriers or for soft tissue augmentation.

[0011] Improved drug formulations of the invention include p-GlcNAcbased materials designed to deliver drugs. These new drug formulationsare an improvement over traditional drug formulations, in that the drugformulations of the invention provide, for example, increasedeffectiveness, reduced toxicity and improved bioavailability. Theseimproved drug formulations can be used in combination with manytherapeutic agents including, but not limited to, chemotherapeuticagents, such as antitumor agents, as well as antibiotics,antibacterials, antifungals and anti-inflammatory drugs.

[0012] Additionally, the present invention relates to cell basedtherapeutics using p-GlcNAc based materials as a matrix for theencapsulation of cells. For example, p-GlcNAc/cell encapsulations may beused for the implantation of insulin-producing cells in the treatment ofdiabetes or dopamine-producing cells for the treatment of Parkinson'sdisease. The p-GlcNAc cell encapsulations of the invention can also beused for the delivery of cells to regenerate tissue such as, forexample, skin, cartilage and bone.

4. BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1. Chemical structure of 100% p-GlcNAc. “n” refers to aninteger ranging from about 4,000 to about 150,000, with about 4,000 toabout 15,000 being preferred.

[0014]FIG. 2. Carbohydrate analysis of p-GlcNAc, Gas Chromatography-MassSpectroscopy data. Solid squares represent p-GlcNAc purified using theacid treatment/neutralization variation of the Chemical/Biologicalmethod, as described in Section 5.3.2, below.

[0015]FIG. 3A. Circular dichroism spectra of solid membranes of purep-GlcNAc.

[0016]FIG. 3B. Circular dichroism spectra of solid membranes ofDeacetylated p-GlcNAc. The disappearance of the 211 nm minimum and 195nm maximum observed in pure p-GlcNAc (FIG. 3A) indicates completedeacetylation under the conditions used, as described in Section 5.4below.

[0017]FIG. 4A. Infra-red spectra analyses of thin membranes of purediatom p-GlcNAc prepared by the mechanical force purification method,top, and the chemical/biological purification method, bottom.

[0018]FIG. 4B. Infra-red spectra analyses of two preparations ofcommercial “chitin” cast into membranes according to the methodsdetailed in Section 5.5, below.

[0019]FIG. 4C. Infra-red spectra analyses of pure p-GlcNAc which wasmodified by heat denaturation (top) and by chemical deacetylation(bottom), according to the methods detailed in Section 5.4, below.

[0020]FIG. 4D. Infra-red spectrum analysis of a p-GlcNAc membranederived from the diatom Thalassiosira fluviatilis, using thechemical/biological purification method, as detailed in Section 5.3.2,below.

[0021]FIG. 4E. Infra-red spectrum analysis of a p-GlcNAc membraneprepared by the mechanical force purification method, as described inSection 5.3.1, below, following autoclaving.

[0022]FIG. 5A. NMR analysis of p-GlcNAc purified using thechemical/biological purification method as described in Section 5.3.2,below. Chart depicting peak amplitudes, areas, and ratios relative toreference controls. Ratio of total areas of peaks.

[0023]FIG. 5B. NMR analysis of p-GlcNAc purified using thechemical/biological purification method as described in Section 5.3.2.The graph depicts the ratios of total areas of peaks.

[0024]FIG. 6. Transmission electron micrographs (TEM) of a p-GlcNAcmembrane prepared by the mechanical force purification method asdescribed in Section 5.3.1, below. Magnification: top, 4190×; bottom,16,250×.

[0025]FIG. 7. Transmission electron micrographs (TEM) of a p-GlcNAcmembrane by HF treatment as described in the discussion of thechemical/biological purification method in Section 5.3.2, below.Magnification: top, 5270×; bottom, 8150×.

[0026]FIG. 8. Transmission electron micrographs (TEM) of a p-GlcNAcmembrane prepared by the acid treatment/neutralization variation of thechemical/biological purification method, as described in Section 5.3.2,below. Magnification: top, 5270×; bottom, 16,700×.

[0027]FIG. 9A. Scanning electron micrograph depicting a p-GlcNAcmembrane prepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 200×.

[0028]FIG. 9B. Scanning electron micrograph depicting a p-GlcNAcmembrane prepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 100×.

[0029]FIG. 9C. Scanning electron micrograph depicting a p-GlcNAcmembrane prepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 5000×.

[0030]FIG. 9D. Scanning electron micrograph depicting a p-GlcNAcmembrane prepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2below. Magnification: 10,000×.

[0031]FIG. 9E. Scanning electron micrograph depicting a p-GlcNAcmembrane prepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 20,000×.

[0032]FIG. 10. Scanning electron micrographs of a pure p-GlcNAc membranemade from material which was initially produced using the celldissolution/neutralization purification method described in Section 5.3,below, dissolved in dimethylacetamide/lithium chloride, andreprecipitated in H₂O into a mat, as described below in Section 5.5.Magnification: top, 1000×, bottom, 10,000×.

[0033]FIG. 11. Scanning electron micrographs of a deacetylated p-GlcNAcmat. Magnification: top, 1000×, bottom, 10,000×.

[0034]FIG. 12. Photographs of diatoms. Note the p-GlcNAc fibersextending from the diatom cell bodies.

[0035]FIG. 13. Diagram depicting some of the possible p-GlcNAc anddeacetylated p-GlcNAc derivatives of the invention. (Adapted from S.Hirano, “Production and Application of Chitin and Chitosan in Japan”, in“Chitin and Chitosan”, 1989, Skjak-Braek, Anthonsen, and Sanford, eds.Elsevier Science Publishing Co., pp. 37-43.)

[0036]FIG. 14. Cell viability study of cells grown in the presence orabsence of p-GlcNAc membranes. Closed circle (): cells grown onp-GlcNAc matrix; open circles (∘): cells grown in absence of matrix.

[0037]FIG. 15. SEM micrographs of transformed mouse fibroblast cellsgrown on p-GlcNAc membranes. Magnification top, 1000×; bottom, 3000×.

[0038]FIG. 16A. Scanning electron micrograph (SEM) of a collagen-onlycontrol material prepared according to the method described, below, inSection 13.1. Magnification 100×.

[0039]FIG. 16B. Scanning electron micrograph (SEM) of acollagen/p-GlcNAc hybrid material prepared according to the methoddescribed, below, in Section 13.1. Ratio collagen suspension:p-GlcNAcsuspension equals 3:1, with final concentrations of 7.5 mg/ml collagenand 0.07 mg/ml p-GlcNAc. Magnification 100×.

[0040]FIG. 16C. Scanning electron micrograph (SEM) of acollagen/p-GlcNAc hybrid material prepared according to the methoddescribed, below, in Section 13.1. Ratio collagen suspension:p-GlcNAcsuspension equals 1:1, with final concentrations of 5.0 mg/ml collagenand 0.12 mg/ml p-GlcNAc. Magnification 100×.

[0041]FIG. 16D. Scanning electron micrograph (SEM) of acollagen/p-GlcNAc hybrid material prepared according to the methoddescribed, below, in Section 13.1. Ratio collagen suspension:p-GlcNAcsuspension equals 2:2, with final concentrations of 10.0 mg/ml collagenand 0.25 mg/ml p-GlcNAc. Magnification 100×.

[0042]FIG. 16E. Scanning electron micrograph (SEM) of acollagen/p-GlcNAc hybrid material prepared according to the methoddescribed, below, in Section 13.1. Ratio collagen suspension:p-GlcNAcsuspension equals 1:3, with final concentrations of 2.5 mg/ml collagenand 0.25 mg/ml p-GlcNAc. Magnification 100×.

[0043]FIG. 17A. SEM of mouse 3T3 fibroblast cells cultured on thecollagen-only control material of FIG. 16A, above. Magnification 100×.

[0044]FIG. 17B. SEM of mouse 3T3 fibroblast cells cultured on thecollagen/p-GlcNAc material of FIG. 16B, abode. Magnification 100×.

[0045]FIG. 17C. SEM of mouse 3T3 fibroblast cells cultured on thecollagen/p-GlcNAc material of FIG. 16C, above. Magnification 100×.

[0046]FIG. 17D. SEM of mouse 3T3 fibroblast cells cultured on thecollagen/p-GlcNAc material of FIG. 16D, above. Magnification 100×.

[0047]FIG. 18. Transformed NMR data curves, used to obtain areas foreach carbon atom and to then calculate the CH3(area) to C-atom(area)ratios.

[0048]FIG. 19. Typical p-GlcNAc C¹³-NMR spectrum. The individual peaksrepresent the contribution to the spectrum of each unique carbon atom inthe molecule.

[0049]FIG. 20. Transformed NMR spectrum data representing valuescalculated for CH3(area) to C-atom(area) ratios. Top: Graphic depictionof data; bottom: numerical depiction of data.

[0050] FIGS. 21A-G. Three-dimensional p-GlcNAc matrices produced invarious solvents. Specifically, the p-GlcNAc matrices were produced indistilled water (FIG. 21A, FIG. 21D), 10% methanol in distilled water(FIG. 21B), 25% methanol in distilled water (FIG. 21C), 10% ethanol indistilled water (FIG. 21E), 25% ethanol in distilled water (FIG. 21F)and 40% ethanol in distilled water (FIG. 21G). Magnification: 200×. Ascale marking of 200 microns is indicated on each of these figures.

[0051] FIGS. 22A-G. Fibroblast cells grown on three-dimensional p-GlcNAcmatrices prepared by lyophilizing p-GlcNAc in distilled water.Magnification: 100× (FIGS. 22A, 22E), 500×(FIG. 22B), 1000× (FIGS. 22C,22F), 5000× (FIGS. 22D, 22G). Scales marking 5, 20, 50, or 200 microns,as indicated, are included in each of the figures.

[0052]FIG. 23. A typical standard curve obtained using the proceduredescribed, below, in Section 18.1. A standard curve such as this one wasused in the lysozyme-chitinase assay also described, below, in Section18.1.

[0053]FIG. 24. p-GlcNAc lysozyme digestion data. The graph presentedhere depicts the accumulation of N-acetylglucosamine over time, asp-GlcNAc membranes are digested with lysozyme. The graph compares thedegradation rate of fully acetylated p-GlcNAc to partially (50%)deacetylated p-GlcNAc, and demonstrates that the degradation rate forthe partially deacetylated p-GlcNAc was substantially higher than thatof the fully acetylated p-GlcNAc material.

[0054]FIG. 25. p-GlcNAc lysozyme digestion data. The graph presentedhere depicts the accumulation of N-acetylglucosamine over tire, asp-GlcNAc membranes are digested with lysozyme. The graph compares thedegradation rate of two partially deacetylated p-GlcNAc membranes(specifically a 25% and a 50% deacetylated p-GlcNAc membrane). The datademonstrate that the degradation rate increases as the percent ofdeacetylation increases, with the degradation rate for the 50%deacetylated p-GlcNAc membrane being substantially higher than that ofthe 25% deacetylated p-GlcNAc membrane.

[0055] FIGS. 26A-26E. p-GlcNAc in vivo biodegradability data.

[0056] FIGS. 26A-26C depict rats which have had prototype 1 (fullyacetylated p-GlcNAc) membrane abdominally implanted, as described,below, in Section 18.1. FIG. 26A shows a rat at day 0 of theimplantation; FIG. 26B shows a rat at day 14 post-implantation; FIG. 26Cshows a rat at day 21 post-implantation. FIGS. 26D-26E depict rats whichhave had prototype 3A (lyophilized and partially deacetylated p-GlcNAcmembrane) abdominally implanted, as described, below, in Section 18.1.FIG. 26D shows a rat at day 0 of the implantation; FIG. 26E shows a ratat day 14 post-implantation.

[0057]FIG. 27. The graph depicted here illustrates data concerning thepercent increase in tumor size of animals which either received notreatment () or received p-GlcNAc-lactate/fluorouracil (5′-FU) (◯), asdescribed, below, in Section 20.1.

[0058]FIG. 28. The graph depicted here illustrates data concerning thepercent increase in tumor size of animals which either receivedp-GlcNAc-lactate alone () or received p-GlcNAc-lactate/fluorouracil(5′-FU) (◯), as described, below, in Section 20.1.

[0059]FIG. 29. The graph depicted here illustrates data concerning thepercent increase in tumor size of animals which either received notreatment () or received p-GlcNAc-lactate/mitomycin (mito) (◯), asdescribed, below, in Section 20.1.

[0060]FIG. 30. The graph depicted here illustrates data concerning thepercent increase in tumor size of animals which either receivedp-GlcNAc-lactate alone () or received p-GlcNAc-lactate/mitomycin (mito)(◯), as described, below, in Section 20.1.

[0061]FIG. 31. The bar graph depicted here illustrates the averagepercent change in tumor size per animal of animals treated withp-GlcNAc/5′-FU high dose (bar 1), p-GlcNAc/5′-FU low dose (bar 2),p-GlcNAc membrane alone (bar 3), and untreated (bar 4). N=4 for bars 1and 2, n=2 for bars 3 and 4.

[0062]FIG. 32A. Photograph of p-GlcNAc-treated site of Yorkshire pigabdomen 21 days after four 2.0×2.0 cm serosal abrasion lesions werecreated.

[0063]FIG. 32B. Photograph of non-treated-control site of Yorkshire pigabdomen 21 days after four 2.0×2.0 cm serosal abrasion lesions werecreated.

5. DETAILED DESCRIPTION OF THE INVENTION

[0064] Presented below, is, first, a description of physicalcharacteristics of the purified p-GlcNAc species of the invention, ofthe p-GlcNAc derivatives, and of their reformulations. Next, methods aredescribed for the purification of the p-GlcNAc species of the inventionfrom microalgae, preferably diatom, starting sources. Third, derivativesand reformulations of the p-GlcNAc, and methods for the production ofsuch derivatives and reformulations are presented. Finally, uses arepresented for the p-GlcNAc, p-GlcNAc derivatives and/or p-GlcNAcreformulations of the invention.

5.1. p-GlcNAc

[0065] The p-GlcNAc polysaccharide species of the invention is a polymerof high molecular weight ranging from a weight average of about 800,000daltons to about 30 million daltons, based upon gel permeationchromatography measurements. Such a molecular weight range represents ap-GlcNAc species having about 4,000 to about 150,000 N-acetylglucosaminemonosaccharides attached in a β-1→4 configuration, with about 4,000 toabout 15,000 N-acetylglucosamine monosaccharides being preferred (FIG.1).

[0066] The variability of the p-GlcNAc of the invention is very low, andits purity is very high, both of which are evidenced by chemical andphysical criteria. Among these are chemical composition andnon-polysaccharide contaminants. First, chemical composition data forthe p-GlcNAc produced using two different purification methods, both ofwhich are described in Section 5.3, below, is shown in Table I below. Ascan be seen, the chemical composition of the p-GlcNAc produced by bothmethods is, within the bounds of experimental error, the same as theformula compositions of p-GlcNAc. Second, as is also shown in Table I,the p-GlcNAc produced is free of detectable protein contaminants, issubstantially free of other organic contaminants such as free aminoacids, and is substantially free of inorganic contaminants such as ashand metal ions (the p-GlcNAc of the invention may deviate up to about 2%from the theoretical values of carbon, hydrogen, nitrogen and oxygen forpure p-GlcNAc). Therefore, as used herein, the terms “substantially freeof organic contaminants” and “substantially free of inorganiccontaminants” refer to compositions of p-GlcNAc having the profiles forcarbon, hydrogen, nitrogen and oxygen which deviate no more than about2% from the theoretical values, and preferably, the p-GlcNAc of theinvention contain a profile as exemplified in the Experimental Data onp-GlcNAc mats in Table I (allowing for the percent deviation). Further,the p-GlcNAc of the invention exhibits a very low percentage of boundwater. TABLE I CHEMICAL ANALYSIS DATA (% by weight) Theoretical Valuesfor Pure p-GlcNAc: Carbon- 47.29 Hydrogen- 6.40 Nitrogen- 6.89 Oxygen-39.41 Protein- 0.00

Experimental Data on p-GlcNAc Mats

[0067] (Number of Experimental Batches for Each Membrane Type BeingGreater Than 30 for Each Membrane Type) MECHANICAL FORCECHEMICAL/BIOLOGICAL METHOD METHOD Normalized ¹ % Dev. Normalized ¹ %Dev. Carbon 47.21 ± 0.08 −0.17 47.31 ± 0.11 +0.04 Hydrogen  6.45 ± 0.08+0.78  6.34 ± 0.08 −0.94 Nitrogen  6.97 ± 0.18 +0.87  6.94 ± 0.16 +0.73Oxygen 39.55 ± 0.36 +0.36 39.41 ± 0.10  0.00 Average Values AverageValues Protein 0.00 0.00 Ash 1.30 0.98 Moisture 2.0  1.2 

[0068] The pure p-GlcNAc of the invention exhibits a carbohydrateanalysis profile substantially similar to that shown in FIG. 2. Theprimary monosaccharide of the pure p-GlcNAc of the invention isN-acetylglucosamine. Further, the pure p-GlcNAc of the invention doesnot contain the monosaccharide glucosamine.

[0069] The circular dichroism (CD) and sharp infra-red spectra (IR) ofthe p-GlcNAc of the invention are shown in FIG. 3A, and FIGS. 4A and 4D,respectively, which present analyses of material produced using themethods described in Section 5.3, below. Such physical data corroboratesthat the p-GlcNAc of the invention is of high purity and crystallinity.The methods used to obtain the CD and IR data are described, below, inthe Working Example in Section 6.

[0070] NMR analysis of the pure p-GlcNAc of the invention exhibits apattern substantially similar to that seen in FIGS. 5A, 5B, 18A and 18B.Such an NMR pattern indicates not only data which is consistent with thep-GlcNAc of the invention being a fully acetylated polymer, but alsodemonstrates the lack of contaminating organic matter within thep-GlcNAc species.

[0071] The electron micrographic structure of the p-GlcNAc of theinvention, as produced using the methods described in Section 5.3, belowand demonstrated in the Working Examples presented, below, in Section 8and 9, is depicted in FIG. 6 through FIG. 9E.

[0072] The p-GlcNAc of the invention exhibits a high degree ofbiocompatability. Biocompatability may be determined by a variety oftechniques, including, but not limited to such procedures as the elutiontest, intramuscular implantation, or intracutaneous or systemicinjection into animal subjects. Briefly, an elution test (U.S.Pharmacopeia XXII, 1990, pp. 1415-1497; U.S. Pharmacopeia XXII, 1991,Supplement 5, pp. 2702-2703) is designed to evaluate thebiocompatability of test article extracts, and assays the biologicalreactivity of a mammalian cell culture line which is sensitive toextractable cytotoxic articles (such as, for example, the L929 cellline) in response to the test article. The Working Example presented inSection 10, below, demonstrates the high biocompatability of thep-GlcNAc of the invention.

5.2. Methods of Producing Microalgal Sources of p-GlcNAc 5.2.1.Microalgal Sources of p-GlcNAc

[0073] The p-GlcNAc of the invention is produced by, and may be purifiedfrom, microalgae, preferably diatoms. The diatoms of several genuses andnumerous species within such genuses may be utilized as p-GlcNAcstarting sources. Each of these diatoms produce p-GlcNAc. See FIG. 12for photographs of such diatoms. The diatoms which may be used asstarting sources for the production of the p-GlcNAc of the inventioninclude, but are not limited to members of the Coscinodiscus genus, theCyclotella genus, and the Thalassiosira genus, with the Thalassiosiragenus being preferred

[0074] Among the Coscinodiscus genus, the species of diatom that may beused to produce the p-GlcNAc of the invention include, but are notlimited to the concinnus and radiatus species. The diatoms among theCyclotella genus which may be used include, but are not limited to thecaspia, cryptica, and meneghiniana species. The Thalassiosira diatomsthat may be utilized to produce the starting material for the p-GlcNAcof the invention include, but are not limited to the nitzschoides,aestivalis, antarctica, deciphens, eccentrica, floridana, fluviatilis,gravida, guillardii, hyalina, minima, nordenskioldii, oceanica,polychorda, pseudonana; rotula, tubifera, tumida, and weissflogiispecies, with the fluviatilis and weissflogii species being preferred.

[0075] Diatoms such as those described above may be obtained, forexample, from the culture collection of the Bigelow Laboratory for OceanSciences, Center for Collection of Marine Phytoplankton (McKown Point,West Boothbay Harbor, Me., 04575).

5.2.2. Methods for Growing Diatoms

[0076] Any of the diatoms described in Section 5.2.1, above, may begrown by utilizing, for example, the methods described in this section.New diatom cultures are initiated by inoculating, under asepticconditions, Nutrient Medium with an aliquot of a mature diatom culture.The Nutrient Medium must be free of all other microorganisms, thereforeall materials, including water, organic components, and inorganiccomponents used in the preparation of the Nutrient Medium must besterile. In addition, it is mandatory that all procedures involved inthis operation be conducted under strictly aseptic conditions, i.e., allcontainers, all transfers of substances from one vessel to another, etc.must be performed in a sterile environment. The quantity of NutrientMedium to be prepared at one time should not exceed what is necessary tostart a new culture. For example, Fernbach flasks which occupyapproximately one square foot of surface may be used as vessels for thediatom cultures, and such vessels require one liter of Nutrient Mediumfor optimum growth of the diatom organism.

[0077] Preparation of the nutrient medium involves the followingoperations:

[0078] a) Acquisition and processing of seawater

[0079] b) Preparation of distilled and deionized water.

[0080] c) Preparation of primary nutrient stocks

[0081] d) Preparation of nutrient working stocks

[0082] e) Preparation of the final nutrient medium

[0083] Filtered seawater may be obtained, for example, from the MarineBiology Laboratory (Woods Hole, Mass.) Seawater containers should bestored at 5° C. (+ or −2° C.) When required, the necessary volume ofwater may be filtered through a Buchner filtration unit, using aSupor-800 polyether sulfone filter membrane with 0.8 micron pore size(Gelman, Inc.). The seawater is then sterilized by autoclaving at, forexample, 121° C. for at least about 15 minutes per liter. On completionof the sterilization process, the capped flasks are immediately cooled,preferably by transfer to a cold room capable of allowing the solutionsto reach a temperature of approximately 5° C. (+ or −2° C.) When it isto be used, solutions are allowed to reach room temperature.

[0084] Tap water is distilled and deionized using standard equipment andprocedures, and collected and stored in clean, securely capped,preferably glass, containers.

[0085] Listed below are formulas which may be followed in preparing thestock solutions necessary for the preparation of the Nutrient Medium. Itis to be understood that while such formulas are to be used as guides,it is intended that routine variations of such formulas which contributeto the preparation of a Nutrient Medium capable of sustaining microalgaldiatom growth sufficient for the p-GlcNAc preparative processesdescribed here also be within the scope of the present invention.

[0086] I. Trace Metal Primary Stocks (TMPS)

[0087] a. 39 mM CuSO₄.5H₂O (copper [II] sulfate pentahydrate) (9.8 gcopper [II] sulfate/L)

[0088] b. 7.5 mM ZnSO₄.7H₂O (Zinc sulfate heptahydrate) (22 g zincsulfate/L)

[0089] c. 42 mM CoCl₂.6H₂O (Cobalt [II] chloride hexahydrate) (10 gcobalt [II] chloride/L)

[0090] d. 91 mM MnCl₂.4H₂O (Manganese [II] chloride tetrahydrate) 18 gmanganese [II] chloride/L)

[0091] e. 26 mM NaMoO₄.2H₂O (Sodium molybdate dihydrate) 6.3 g sodiummolybdate/L)

[0092] f. 1 mM H₂SeO₃ (Selenious acid) (0.129 g selenious acid/L).

[0093] Sterile filter each nutrient with a filter of no greater than 0.2mm pore size.

[0094] II Vitamin Primary Stocks (VPS)

[0095] a. 1 mg/ml Vitamin B12

[0096] b. 0.1 mg/ml Biotin

[0097] Sterile filter both stocks with a filter of no greater than 0.2mm pore size.

[0098] III. Sodium Salts Working Stocks (SSWS)

[0099] a. Sodium nitrate working stock: 0.88 M (75 g NaNO₃/L)

[0100] b. Sodium phosphate monobasic monohydrate working stock: 36.2 mMNaH₂PO₄.H₂O (5 g NaH₂PO₄H₂O/L)

[0101] c. Sodium metasilicate monohydrate working stock: 0.11 MNa₂SiO₃.9H₂O (30 g Na₂SiO₃.9H₂O/L)

[0102] Sterile filter each of the SSWS with a filter of no greater than0.2 mm pore size.

[0103] IV. Trace Metal Working Stocks (TMWS)

[0104] 11.7 mM Na₂EDTA (Ethylenediamine Tetraacetic acid, disodium saltdihydrate) (4.36 g/L)

[0105] 11.7 mM FeCl₃.6H₂O (Iron [III] chloride hexahydrate) (3.15 g/L)

[0106] 1 ml/L of each of the six primary trace metal stocks listedabove.

[0107] Sterile filter with a filter of no greater than 0.2 mm pore size.Note that the trace metal working stock must be prepared fresh weekly.

[0108] V. Vitamin Working Stock (VWS)

[0109] 1.0 μg/ml Biotin (1.0 ml primary Biotin Stock/100 ml)

[0110] 1.0 μg/ml Vitamin B12 (0.1 ml Vitamin B12 primary stock/100 ml)

[0111] 20 mg of Thiamine HCl (Thiamine hydrochloride/100 ml).

[0112] Sterile filter with a filter of no greater than 0.2 mm pore size.Note that a new Vitamin Working Stock should be prepared fresh weekly.

[0113] Described below are techniques which may be followed for thepreparation of Nutrient Medium and for diatom culturing. It is to beunderstood that, in addition to these techniques, any routine variationin the formulas and/or procedures described herein which result in aNutrient Medium and in procedures capable of sustaining diatom growthsufficient for the preparative processes described herein is intended tobe within the scope of the present invention.

[0114] Nutrient Medium may be prepared, for example, as follows: To eachliter of filtered and sterilized seawater may be added 1 ml of the NaNO₃working stock, 1 ml of the NaH₂PO4.H₂O working stock, 1 ml of the TraceMetal working stock, and 1 ml of the Na₂SiO₃.9H₂O working stock.Simultaneously with the addition of Na₂SiO₃.9H₂O, 2 mls of 1 N HCl maybe added and the solution may be shaken to mix. Next, 1.5 mls 1 N NaOHmay be added and the solution may again be shaken to mix. Finally, 0.5ml of the Vitamin working stock may be added.

[0115] In order to grow a new diatom culture, 7 ml of a mature culture,(having a cell density within a range of about 1×10⁵ to about 1×10⁶cells/ml.), may be transferred to a sterile container containing 100 mlof sterile Nutrient Medium, which may be prepared according to themethods described above. The inoculated culture may then be incubatedfor 8 days under the following conditions:

[0116] Temperature: 20 degrees Centigrade

[0117] Constant illumination.

[0118] Agitation: Gentle swirling of flasks once per day.

[0119] After 8 days of incubation, 80 ml of this incubated culture maybe transferred, under sterile conditions, to 1000 ml of Nutrient Medium,which may, for example, be contained in a 2.8 L Fernbach flask,protected by a cotton wool plug covered by cheesecloth. Such a culturemay be allowed to incubate and grow to the desired cell density, oralternatively, may be used to inoculate new diatom cultures. Once aculture reaches a desired cell density, the culture's p-GlcNAc fibersmay be harvested, and the p-GlcNAc of the invention may be purified,using methods such as those described below in Section 5.3, below.

[0120] CO₂ may be dissolved in the culture solution in order to maintaina culture pH of approximately 7 to 8, with approximately 7.4 beingpreferred. The maintenance of such a neutral pH environment, greatlyincreases the p-GlcNAc yield that may be obtained from each diatomculture.

5.3. Methods for Isolation, Purification, and Concentration of p-GlcNAcFibers

[0121] Presented in this Section are methods which may be utilized forthe preparation of p-GlcNAc fibers from diatom cultures such as thosedescribed, above, in Section 5.2.

[0122] While each of the methods described below for the purification ofp-GlcNAc from microalgae, preferably diatom, starting sources producesvery pure, unadulterated, crystalline p-GlcNAc, each of the methodsyields p-GlcNAc having specific characteristics and advantageousfeatures. For example, the p-GlcNAc of the invention purified via theMechanical Force method presented in Section 5.3.1, below, produces ap-GlcNAc membrane that provides a superior substrate for the attachmentof cells to the p-GlcNAc. The second method, described below in Section5.3.2, the Chemical/Biological method, produces a much higher averageyield than the average p-GlcNAc yield produced by the Mechanical Forcemethod. Additionally, the acid treatment/neutralization variationdescribed as part of the Chemical/Biological method of Section 5.3.2,below, produces extremely long p-GlcNAc fibers, with some fibers beingin excess of 100 μm, and containing molecules of the p-GlcNAc polymer ofvery high molecular weight, as high as 20-30 million daltons.

5.3.1. Mechanical Force Method for Preparation of Pure p-GlcNAc

[0123] The p-GlcNAc fibers may be separated from diatom cell bodies bysubjecting the contents of the culture to an appropriate mechanicalforce. Such a mechanical force may include, but is not limited to, ashear force generated by, for example, a colloid mill, an ultrasounddevice, or a bubble generator, or a cutting force generated by, forexample, a Waring blender.

[0124] The resulting suspension of diatom cell bodies and p-GlcNAcfibers are then segregated. For example, the suspension may be subjectedto a series of centrifugation steps which segregate the p-GlcNAc fibersfrom the cell bodies, yielding a clear supernatant exhibiting little, ifany, visible flocculent material. A fixed angle rotor, and a temperatureof about 10° C. are preferred for the centrifugation steps. The speed,duration, and total number of centrifugation steps required may varydepending on, for example, the specific centrifugation rotor being used,but the determination of the values for such parameters will be apparentto one of ordinary skill in the art.

[0125] The p-GlcNAc fibers in the supernatant may then be concentratedusing techniques well known to those of skill in the art. Suchtechniques may include, but are not limited to suction and filtrationdevices.

[0126] Finally, the concentrated p-GlcNAc fibers are washed with, forexample, distilled-deionized water, HCl and ethanol, or otherappropriate solvents, preferably solvents, such as alcohols, in whichboth organic and inorganic materials dissolve.

[0127] The Working Example presented in Section 7, below, demonstratesthe use of this method for the purification of p-GlcNAc.

5.3.2. Chemical/Biological Method for Purification of p-GlcNAc

[0128] In this method, p-GlcNAc fibers are separated from diatom cellbodies by subjecting them to chemical and/or biological agents asdescribed in more detail below.

[0129] Diatom cultures may be treated with a chemical capable ofweakening diatom cell walls, which leads to a release of the p-GlcNAcfibers without altering their structure. Such a chemical may include,but is not limited to, hydrofluoric acid (HF). Alternatively, a maturediatom culture may be treated with a biological agent capable ofaltering a biological process may be used to inhibit p-GlcNAc fibersynthesis, thus releasing the fibers already present. For example, suchan agent may include, but is not limited to, polyoxin-D, an inhibitor ofthe enzyme N-acetylglucosaminyl-P-transferase.

[0130] The cell bodies and p-GlcNAc-containing fibers of diatom culturestreated with a member of the above described chemical or biologicalagents are then segregated. For example, the contents of treated diatomcultures may be allowed to settle such that the contents of the culturesare allowed to form two distinct layers. The upper layer will containprimarily the p-GlcNAc fibers, while the bottom layer will contain thecell bodies. The upper p-GlcNAc fiber-containing layer may be siphonedoff, leaving behind the settled cellular material of the bottom layer.

[0131] The siphoned off p-GlcNAc fiber-containing layer may then befurther purified to remove protein and other unwanted matter bytreatment with a detergent that will not damage the p-GlcNAc fibers.Such a detergent may include, but is not limited to, sodium dodecylsulfate (SDS).

[0132] When acid treatment, such as HF treatment, is used to separatep-GlcNAc fibers from diatom cell bodies, a step may be included for thedispersal of the fibers. Such a step may include, but is not limited to,the use of mechanical force for fiber dispersal, such as a step in whichthe fibers are subjected to the movements of an orbital shaker.

[0133] Alternatively, the acid-treated suspension may, in an optionalstep, be neutralized prior to further purification by detergenttreatment. Such neutralization will, in general, change the pH of thesuspension from approximately 1.8 to approximately 7.0, and may beaccomplished by, for example, the addition of an appropriate volume of1M Tris (pH 8.0) or the addition of an appropriate volume of sodiumhydroxide (NaOH) Neutralization, in general, yields pure p-GlcNAc fibersof a substantially greater length than the other purification methodsdiscussed herein.

[0134] The purified p-GlcNAc fibers may then be concentrated usingtechniques well known to those of skill in the art, such as by utilizinga suction and filtration device. Finally, the p-GlcNAc fibers arewashed, in a series of steps with distilled-deionized water, Cl andethanol, or other appropriate solvents, preferably solvents, such asalcohols, in which both organic and inorganic materials dissolve.

[0135] The Working Example presented, below, in Section 8 demonstratesthe successful utilization of such a purification method.

5.4. Derivatization of p-GlcNAc

[0136] The pure, fully acetylated p-GlcNAc of the invention may bederivatized, by utilizing a variety of controlled conditions andprocedures, into a large range of different compounds. See FIG. 13 for adiagram depicting some of these compounds. Such derivatized compoundsmay include, but are not limited to, partially or completelydeacetylated p-GlcNAc, which has been modified via chemical and/orenzymatic means, as described in further detail, below. Additionally,p-GlcNAc, or its deacetylated derivative, may be derivatized by beingsulfated, phosphorylated, and/or nitrated. Further, as detailed below,O-sulfonyl, N-acyl, O-alkyl, N-alkyl, deoxyhalogen, and N-alkylidene andN-arylidene and other derivatives may be prepared from the p-GlcNAc ordeacetylated p-GlcNAc of the invention. The deacetylated p-GlcNAc of theinvention may also be used to prepare a variety of organic salts and/ormetal chelates. Further, the p-GlcNAc, or a derivative thereof, of theinvention may have attached to it, either covalently or non-covalently,any of a variety of molecules. Still further, the p-GlcNAc of theinvention, or a derivative thereof, may be subjected to controlledhydrolysis conditions which yield groups of molecules having uniform anddiscrete molecular weight characteristics.

[0137] One or more of the monosaccharide units of the p-GlcNAc of theinvention may be deacetylated to form a poly-β-1→4-N-glucosaminespecies. A poly-β-1→4-N-glucosamine species of the invention in whicheach of the monosaccharide units of the poly-β-1→4-N-acetylglucosaminespecies of the invention has been deacetylated will have a molecularweight of about 640,000 daltons to about 24 million daltons, with about640,000 daltons to about 2.4 million daltons being preferred. A specieswith such a molecular weight range represents a species having about4000 to about 150,000 glucosamine monosaccharides covalently attached ina β-1→4 configuration, with about 4,000 to about 15,000 glucosaminemonosaccharides being preferred. At least one of the monosaccharideunits of the poly-β-1→4-N-glucosamine species may remain acetylated,with about 25% to about 75% acetylation being preferred, and about 30%acetylation being most preferred.

[0138] The p-GlcNAc of the invention may be deacetylated by treatmentwith a base to yield glucosamines with free amino groups. Thishydrolysis process may be carried out with solutions of concentratedsodium hydroxide or potassium hydroxide at elevated temperatures. Toprecisely control the extent of deacetylation and to avoid degradationof the main carbohydrate chain of the polysaccharide molecule, however,it is preferable that an enzymatic procedure utilizing a chitindeacetylase enzyme be used for p-GlcNAc deacylation. Such a deacetylaseenzymatic procedure is well known to those of skill in the art and maybe performed as in (U.S. Pat. No. 5,219,749), which is incorporatedherein, by reference, in its entirety.

[0139] One or more of the monosaccharide units of the p-GlcNAc of theinvention may be derivatized to contain at least one sulfate group, or,alternatively, may be phosphorylated or nitrated, as depicted below:

[0140] where, R and/or R₁, in place of a hydrogen, and/or R₂, in placeof —COCH₃, may be a sulfate (—SHO₃), a phosphate (—P(OH)₂), or a nitrate(—NO₂) group.

[0141] Described below are methods by which such p-GlcNAc derivativesmay be prepared. Before performing methods such as those described inthis Section, it may be advantageous to first lyophilize, freeze inliquid nitrogen, and pulverize the p-GlcNAc starting material.

[0142] Sulphated p-GlcNAc derivatives may be generated, by, for example,a two step process. In the first step, O-carboxymethyl p-GlcNAc may beprepared from the p-GlcNAc and/or p-GlcNAc derivatives of the inventionby, for example, utilizing techniques such as those described by Tokuraet al. (Tokura, S. et al., 1983, Polym. J. 15:485. Second, the sulfationstep may be carried out with, for example, N,N-dimethyl-formamide-sulfurtrioxide, according to techniques well known to those of skill in theart, such as are described by Schweiger (Schweiger, R. G., 1972,Carbohydrate Res. 21:219). The resulting product may be isolated as asodium salt.

[0143] Phosphorylated p-GlcNAc derivatives of the invention may beprepared, for example, by utilizing techniques well known to those ofskill in the art, such as those described by Nishi et al. (Nishi, N. etal., 1986, in “Chitin in Nature and Technology, Muzzarelli et al., eds.Plenum Press, New York, pp. 297-299). Briefly, p-GlcNAc/methanesulfonicacid mixture may be treated with phosphorus pentoxide (in anapproximately 0.5 to 4.0 molar equivalent) with stirring, at atemperature of about 0° C. to about 5° C. Treatment may be for about 2hours. The resulting product may then be precipitated and washed usingstandard techniques well known to those of skill in the art. Forexample, the sample may be precipitated with a solvent such as ether,centrifuged, washed with a solvent such as ether, acetone, or methanol,and dried.

[0144] Nitrated p-GlcNAc derivatives may be prepared by utilizingtechniques well known to those of skill in the art, such as thosedescribed by Schorigin and Halt (Schorigin, R. and Halt, E., 1934, Chem.Ber. 67:1712). Briefly, p-GlcNAc and/or a p-GlcNAc derivative may betreated with concentrated nitric acid to form a stable nitrated product.

[0145] One or more of the monosaccharide units of the p-GlcNAc of theinvention may contain a sulfonyl group, as depicted below:

[0146] where R₃ may be an alkyl, an aryl, an alkenyl, or an alkynylmoiety. Such a derivative may be generated by well known methods such asthe method described in Kurita et al. (Kurita, K. et al., 1990, Polym.Prep [Am. Chem. Soc., Div. Polym. Chem.] 31:624-625). Briefly, anaqueous alkali p-GlcNAc solution may be reacted with a chloroformsolution of tosyl chloride, and the reaction may then be allowed toproceed smoothly at low temperatures.

[0147] One or more of the monosaccharides of the p-GlcNAc of theinvention or its deacetylated derivative may contain one or more O-acylgroups, as depicted below:

[0148] where R₄ and/or R₅, in place of hydrogen, may be an alkyl, analkenyl, or an alkynyl moiety, and R₆ may be an alkyl, an alkenyl, or analkynyl moiety. An example of such a derivative may be generated by wellknown methods such as those described by Komai (Komai, T. et al., 1986,in “Chitin in Nature and Technology”, Muzzarelli et al., eds., PlenumPress, New York, pp. 497-506). Briefly, p-GlcNAc may be reacted with anyof a number of suitable acyl chlorides in methanesulfonic acid to yieldp-GlcNAc derivatives which include, but are not limited to, caproyl,capryl, lanoyl, or benzoyl derivatives.

[0149] One or more of the monosaccharides of the deacetylated p-GlcNAcof the invention may contain an N-acyl group, as depicted below:

[0150] where R₇ may be an alkyl, an alkenyl, or an alkynyl moiety. Sucha derivatization may be obtained by utilizing techniques well known tothose of skill in the art, such as the technique described in Hirano etal. (Hirano, S. et al., 1976, Carbohydrate Research 47:315-320).

[0151] Deacetylated p-GlcNAc is soluble in a number of aqueous solutionsof organic acids. The addition of selected carboxylic anhydrides to suchp-GlcNAc-containing solutions, in aqueous methanolic acetic acid,results in the formation of N-acyl p-GlcNAc derivatives.

[0152] One or more of the monosaccharides of the deacetylated p-GlcNAcof the invention or of its deacetylated derivative, may contain anO-alkyl group, as depicted below:

[0153] where R₈ may be an alkyl, and alkenyl, or a alkynyl moiety. Sucha derivatization may be obtained by using techniques well known to thoseof skill in the art. For example, the procedure described by Maresh etal. (Maresh, G. et al., in “Chitin and Chitosan,” Skjak-Braek, G. etal., eds., 1989, Elsevier Publishing Co., pp. 389-395). Briefly,deacetylated p-GlcNAc may be dispersed in dimethoxyethane (DME) andreacted with an excess of propylene oxide. The period of the reactionmay be 24 hours, and the reaction takes place in an autoclave at 40 to90° C. The mixture may then be diluted with water and filtered. The DMEmay be removed by distillation. Finally, the end-product may be isolatedvia lyophilization.

[0154] One or more of the monosaccharide units of the p-GlcNAc of theinvention may be an alkali derivative, as depicted below:

[0155] Such a derivative may be obtained by using techniques well knownto those of skill in the art. For example, a method such as thatdescribed by Noguchi et al. (Noguchi, J. et al., 1969, Kogyo KagakuZasshi 72:796-799) may be utilized. Briefly, p-GlcNAc may be steeped,under vacuo, in NaOH (43%, preferably) for a period of approximately twohours at about 0° C. Excess NaOH may then be removed by, for example,centrifugation in a basket centrifuge and by mechanical pressing.

[0156] One or more of the monosaccharide units of the deacetylatedderivative of the p-GlcNAc of the invention may contain an N-alkylgroup, as depicted below:

[0157] where R₉ may be an alkyl, an alkenyl, or an alkynyl moiety. Sucha derivatization may be obtained by utilizing, for example, a proceduresuch as that of Maresh et al. (Maresh, G. et al., in “Chitin andChitosan,” Skjak-Brack, G. et al., eds. 1989, Elsevier Publishing Co.,pp. 389-395), as described, above, for the production of O-alkylp-GlcNAc derivatives.

[0158] One or more of the monosaccharide units of the deacetylatedderivative of the p-GlcNAc of the invention may contain at least onedeoxyhalogen derivative, as depicted below:

[0159] where R₁₀ may be F, Cl, Br, or I, with I being preferred. Such aderivative may be obtained by using techniques well known to those ofskill in the art. For example, a procedure such as that described byKurita et al. (Kurita, K. et al., 1990, Polym. Prep. [Am. Chem. Soc.Div. Polym. Chem.] 31:624-625) may be utilized. Briefly, a tosylatedp-GlcNAc is made to react with a sodium halide in dimethylsulfoxide,yielding a deoxyhalogen derivative. p-GlcNAc tosylation may be performedby reacting an aqueous alkali p-GlcNAc solution with a chloroformsolution of tosyl chloride. Such a reaction may proceed smoothly at lowtemperatures.

[0160] One or more of the monosaccharide units of the deacetylatedderivative of the p-GlcNAc of the invention may form a salt, as depictedbelow:

[0161] where R₁₁ may be an alkyl, an alkenyl, or an alkynyl moiety. Sucha derivatization may be obtained by using techniques well known to thoseof skill in the art. For example, a procedure such as that described byAustin and Sennett (Austin, P. R. and Sennett, S., in “Chitin in Natureand Technology,” 1986, Muzzarelli, R. A. A. et al., eds. Plenum Press,pp. 279-286) may be utilized. Briefly, deacetylated p-GlcNAc may besuspended in an organic medium such as, for example, ethyl acetate orisopropanol, to which may be added an appropriate organic acid such as,for example, formic, acetic, glycolic, or lactic acid. The mixture maybe allowed to stand for a period of time (1 to 3 hours, for example) Thetemperature of reaction and drying may vary from about 12° to about 35°C., with 20° to 25° C. being preferred. The salts may then be separatedby filtration, washed with fresh medium, and the residual mediumevaporated.

[0162] One or more of the monosaccharide units of the deacetylatedderivative of the p-GlcNAc of the invention may form a metal chelate, asdepicted below:

[0163] where R₁₂ may be a metal ion, particularly one of the transitionmetals, and X is the dative bond established by the nitrogen electronspresent in the amino and substituted amino groups present in thedeacetylated p-GlcNAc.

[0164] One or more of the monosaccharide units of the deacetylatedderivative of the p-GlcNAc of the invention may contain an N-alkylideneor an N-arylidene group, as depicted below:

[0165] where R₁₃ may be an alkyl, an alkenyl, an alkynyl, or an arylmoiety. Such a derivatization may be obtained by using techniques wellknown to those of skill in the art. For example, a procedure such asthat described by Hirano et al. (Hirano, S. et al., 1981, J. Biomed.Mat. Res. 15:903-911) may be utilized. Briefly, an N-substitutionreaction of deacetylated p-GlcNAc may be performed with carboxylicanhydrides and/or arylaldehydes to yield acyl- and/or arylidenederivatives.

[0166] Further, the p-GlcNAc of the invention, or its deacetylatedderivative, may be subjected to controlled hydrolysis conditions, whichyield groups of molecules having uniform, discrete molecular weight andother physical characteristics. Such hydrolysis conditions may include,for example, treatment with the enzyme, lysozyme. p-GlcNAc may beexposed to lysozyme for varying periods of time, in order to control theextent of hydrolysis. In addition, the rate of hydrolysis may becontrolled as a function of the extent to which the p-GlcNAc that isbeing lysozyme treated has been deacetylated. Deacetylation conditionsmay be as described earlier in this Section. The more fully a p-GlcNAcmolecule has been deacetylated, between about 20 and about 90 percentdeacetylated, the more fully the molecule will be hydrolyzed in a giventime. Changes in physical characteristics, in addition to the loweringof molecular weight, may be elicited by hydrolysis and/or deacetylationtreatments. Extensive hydrolysis causes liquefication of the p-GlcNAc.The results of a hydrolysis/deacetylation procedure are presented belowin the Working Example of Section 9, below.

[0167] Further, heat denaturation may function to modify the crystallinestructure of the p-GlcNAc. Such a modification of the p-GlcNAc productcrystalline structure may advantageously affect, for example, thereactivity of the p-GlcNAc.

[0168] Further, a variety of molecules may be covalently ornon-covalently functionally attached to the deacetylated derivatives ofthe p-GlcNAc of the invention. Such molecules may include, but are notlimited to such polypeptides as growth factors, such as nerve growthfactor, proteases, such as pepsin, hormones, or peptide recognitionsequences such as RGD sequences, fibronectin recognition sequences,laminin, integrins, cell adhesion molecules, and the like. See, e.g.,the compounds discussed, below, in Section 5.6.1.1. Covalent attachmentof molecules to the exposed primary amines of deacetylated p-GlcNAc maybe accomplished by, for example, chemical attachment utilizingbi-functional cross-linking reagents that act as specific lengthchemical spacers. Such techniques are well known to those of skill inthe art, and may resemble, for example, the methods of Davis and Preston(Davis, M. and Preston, J. F. 198′, Anal. Biochem. 116:404-407) andStaros et al. (Staros, J. V. et al., 1986, Anal. Biochem. 156:220-222).Briefly, carboxylic residues on the peptide to be attached to thedeacetylated or partially deacetylated p-GlcNAc of the invention may beactivated and then crosslinked to the p-GlcNAc. Activation may beaccomplished, for example, by the addition of a solution such ascarbodiimide EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to apeptide solution in a phosphate buffer. Preferably, this solution wouldadditionally contain a reagent such assulpho-NHS(N-hydroxysulphosuccinimide) to enhance coupling. Theactivated peptide may be crosslinked to the deacetylated p-GlcNAc bymixing in a high pH buffer, such as carbonate buffer (pH 9.0-9.2).

[0169] The biological activity of the attached peptide (or anycovalently attached molecule) can be maintained by varying the length ofthe linker molecule (e.g., the bi-functional crosslinking compound)utilized to attach the molecule to the

[0170] p-GlcNAc of the invention. An appropriate linker length for agiven molecule to be attached which will not alter the biologicalactivity of the attached molecule can routinely be ascertained. Forexample, the biological, activity (e.g., a therapeutically effectivelevel of biological activity) of a molecule which has been attached viaa linker of a given length can be tested by utilizing well-known assaysspecific for the given molecule being attached.

[0171] Additionally, in order to maintain the biological activity of themolecule being attached, it may be necessary to utilize a linker whichcan be cleaved by an appropriate naturally occurring enzyme to releasethe peptide (or any covalently attached molecule).

[0172] As above, assays commonly employed by those of skill in the artmay be used to test for the retention of the biological activity of theparticular molecule being attached to ensure that an acceptable level ofactivity (e.g., a therapeutically effective level activity) is retained.

[0173] Alternatively, molecules such as those described above may benon-covalently attached to p-GlcNAc and its derivatives using techniqueswell known to those of skill in the art. For example, a molecule ormolecules of choice may be mixed with suspensions of p-GlcNAc, withdeacetylated or partially deacetylated p-GlcNAc solution, with ap-GlcNAc-lactate solution, with a deacetylated or partially deacetylatedp-GlcNAc salt solution, or with any p-GlcNAc derivative solution. Themixtures can then be lyophilized. Molecules become bound to the p-GlcNAcmatrices following lyophilization, presumably via hydrophobic,electrostatic and other non-covalent interactions. Such p-GlcNAcformulations are, therefore, very easy to produce. Further, suchformulations can effectively be achieved with a wide variety ofmolecules having a broad spectrum of physical characteristics and watersolubility properties, ranging from the most hydrophobic to the mosthydrophilic. Upon attachment of the molecule or molecules, assayscommonly employed by those of skill in the art to test the activity ofthe particular non-covalently attached molecule or molecules can be usedto ensure that an acceptable level of activity (e.g., a therapeuticallyeffective activity) is achieved with the attached molecule.

[0174] Encapsulation using the p-GlcNAc of the invention may be achievedusing methods known in the art. For example, one method for achievingthe p-GlcNAc encapsulation can involve the procedure outlined by Hwanget al. (Hwang, C. et al. in Muzzarelli, R. et al., eds., 1985, “Chitinin Nature and Technology”, Plenum Press, pp. 389-396) which isincorporated by reference in its entirety.

[0175] Encapsulation can also be achieved, for example, by following amodification of the acid treatment/neutralization variation of thechemical/biological purification method presented, above, in Section5.3.2. Rather than raising the pH of the p-GlcNAc solution toapproximately neutral pH range (i.e., approximately 7.4), one may createa basic pH environment, by raising the pH to approximately 9.0 after thepurification of the p-GlcNAc is completed. At a more basic pH, thestructure of the p-GlcNAc of the invention, or a derivative thereof,assumes a more three-dimensional or “open” configuration. As the pH islowered, the molecule's configuration reverts to a more compact,“closed” configuration. Thus, a compound or drug of interest may beadded to a p-GlcNAc at a high pH, then the pH of the p-GlcNAc/drugsuspension may be lowered, thereby “trapping” or encapsulating the drugof interest within a p-GlcNAc matrix. Alternatively, hybrids comprisingp-GlcNAc and/or p-GlcNAc derivatives may be formed. Such hybrids maycontain any of a number of natural and/or synthetic materials, inaddition to p-GlcNAc and/or p-GlcNAc derivatives. For example, hybridsmay be formed of p-GlcNAc and/or p-GlcNAc derivatives plus one or moreextracellular matrix (ECM) components. Such ECM components may include,but are not limited to, collagen, fibronectin, glycosaminoglycans,and/or peptidoglycans. Hybrids may also be formed of p-GlcNAc and/orp-GlcNAc derivatives plus one or more synthetic materials such as, forexample, polyethylene. Such a p-GlcNac/polyethylene or p-GlcNacderivative/polyethylene hybrid may be made by thermally linking thehybrid components via, for example, autoclaving.

[0176] In the case of a collagen/p-GlcNAc hybrid, briefly, a p-GlcNAcsuspension and a collagen suspension may be mixed and lyophilized, andcrosslinked, preferably dehydrothermally crosslinked. The collagenspecies of such hybrids may be native or synthetic, and may be of humanor non-human, such as bovine, for example, origin. p-GlcNAc/collagenand/or p-GlcNAc derivative/collagen hybrid materials exhibit uniformproperties, and form a porous matrix that may act, for example, as anefficient three-dimensional matrix for the attachment and growth ofcells. The Working Example presented in Section 13, below demonstratesthe formation, properties and usefulness of such a p-GlcNAc/collagenhybrid.

[0177] Additionally, an iodo-p-GlcNAc derivative may be copolymerizedwith, for example, styrene, for the manufacture of novel plasticmaterials. Iodo-p-GlcNAc can be prepared by a process similar to thatdescribed by Kurita and Inoue (Kurita, K. and Inoue, S., 1989, in“Chitin and Chitosan”, Skjak-Braek et al., eds., Elsevier SciencePublishing Co., Inc., p. 365), via-tosylation and iodination ofp-GlcNAc. The iodo derivative of p-GlcNAc can then be dispersed innitrobenzene and reacted with styrene, with tin (IV) chloride being usedas a catalyst.

[0178] Hybrids comprising combinations of deacetylated p-GlcNAc and suchcompounds as, for example, heparin, sodium alginate, and carboxymethylp-GlcNAc may be formulated using techniques such as those describedherein. Such combinations may be formed or reformed into, for example,membranes and fibers.

[0179] Complexes of deacetylated p-GlcNAc with polyanions such as, forexample, polyacrylic acid or pectin, possessing both positive andnegative charges, may be formulated. The formation of such complexes maybe accomplished according to a method similar to that described byMireles et al. (Mireles, C. et al., 1992, in “Advances in Chitin andChitosan”, Brine, C. J. et al., eds., Elsevier Publishers, Ltd.).Deacetylated p-GlcNAc and polyacrylic acid, carrageenan or pectin, forexample, are dissolved in HCl and NaCl, respectively, and the reactantsolutions, with equal pH, are mixed. This operation produces effectivemolecules possessing both positive and negative characteristics, useful,for example, in the immobilization of enzymes and therapeutic compounds.

[0180] Further, derivatives, such as partially deacetylated derivativesand carboxymethyl derivatives of the p-GlcNAc of the invention, can beused to coat liposomes to give them greater stability. Liposomes areartificially constructed microspheres of lipid bilayer useful, forexample, for drug delivery. The compositions of present invention can beused to form negatively charged phospholipids bound to positivelycharged deacetylated p-GlcNAc enclosing an aqueous compartment. Theseconstructions are designed to contain and deliver pharmaceuticals orother components more efficiently than would otherwise be possible byway of oral or other application. Such constructions can be produced byutilizing the pGlcNAc compositions of the invention in conjunction withmethods well known in the art. See, for example, the Dong and Rogers(Dong, C. and Rogers, J. A., 1991, Journal of Controlled Release,17:217-224) which is incorporated by reference in its entirety.

[0181] Certain derivatizations of the p-GlcNAc of the invention, or ofits derivatives, may be preferred for specific applications, which aredescribed in Section 5.6, below. For example, sulfated, phosphorylated,and/or nitrated p-GlcNAc derivatives may be preferred as anticoagulantsor as lipoprotein lipase activators. N-acyl p-GlcNAc derivatives mayalso be preferred for anticoagulants, in addition to being preferredfor, for example, use in production of artificial blood vessels,anti-viral compounds, anti-tumor (specifically, cancer cell aggregatingcompounds), dialysis and ultrafiltration membranes, and in theproduction of controlled release drug delivery systems. O-alkyl p-GlcNAcand its deacetylated derivatives may also be preferred in the productionof controlled release drug delivery systems. N-alkyl p-GlcNAcderivatives may be preferred as anti-bacterial agents. Oxido deaminatedderivatives may be preferred as anti-cancer agents, specifically theiruse in conjunction with immunotherapy for cancer cells. Deacetylatedp-GlcNAc derivatives may be preferred as wound healing agents.N-alkylidene and N-arylidene p-GlcNAc derivatives may be preferred forthe enzyme immobilization applications.

5.5. Reformulations

[0182] The p-GlcNAc of the invention, as well as its deacetylatedderivatives and/or their derivatizations, such as those described,above, in Section 5.4, may be dissolved and subsequently reformulatedinto a variety of shapes and configurations.

[0183] Solution of the p-GlcNAc of the invention can be achieved bytreatment with dimethyl acetamide (DMA)/lithium chloride. p-GlcNAc maybe readily dissolved by stirring in a DMA solution containing 5% LiCl(by weight of the DMA). Water soluble p-GlcNAc derivatives, such asp-GlcNAc salts, may be dissolved in water. P-GlcNAc which has been atleast about 75% deacetylated may be put into solution in, for example, amild acidic solution, such as 1 acetic acid. p-GlcNAc derivatives thatare water-insoluble may be put into solution in organic solvents.

[0184] Derivatization of p-GlcNAc in DMA:LiCl with phenyl isocyanatesmay be used to produce carbanilates. Further, derivatization of p-GlcNAcin DMA:LiCl with toluene-p-sulphonylchloride may be used to producetoluene-p-sulfonate.

[0185] The p-GlcNAc of the invention, its deacetylated derivatives,and/or their derivatizations in solution may then be precipitated andreformulated into shapes which include, but are not limited to, mats,strings, microspheres, microbeads, membranes, fibers, powders, andsponges. Further, ultrathin (i.e., less than about 1 micron thick)uniform membranes may be formulated. Additionally, pharmaceuticalformulations such as pills, tablets and capsules can be prepared.

[0186] Such reformulations may be achieved, by, for example, takingadvantage of the fact that pure p-GlcNAc is insoluble in solutions suchas water and alcohol, preferably ethanol. Introduction, by conventionalmeans, such as by injection, for example, of the p-GlcNAc-containingDMA/LiCl mixture into such a water or alcohol, preferably ethanol,solution will bring about the reprecipitation, and thereforereformulation, of the dissolved p-GlcNAc. Such a pure p-GlcNAcreformulation is demonstrated in the Working Example presented, below,in Section 11. In the case of water soluble p-GlcNAc derivatives,reformulations may be achieved by reprecipitating in such organicsolvents as, for example, ethyl acetate or isopropanol. Reformulationsof p-GlcNAc which has been at least about 75% deacetylated may beachieved by reprecipitating in an alkaline solution. Water-insolublep-GlcNAc derivatives may be reformulated by reprecipitation in aqueoussolutions, such as, for example, water.

[0187] Deacetylated p-GlcNAc, in conjunction with oxidized cotton, maybe formulated to produce p-GlcNAc/cotton hybrid materials improving thewet-strength of paper products. An oxidized cotton substrate can beapproached closely by the deacetylated p-GlcNAc chain which has a flatribbon-like shape, similar to that of cellulose. Such proximitymaximizes the contribution of the van der Waals forces to the forcespromoting adsorption, thus enhancing the wet-strength properties of thehybrid p-GlcNAc-cellulose materials. p-GlcNAc membranes andthree-dimensional p-GlcNAc matrices may be produced via methods whichprovide for the formation of controlled average pore sizes within eitherthe membranes or the matrices. Pore size can be controlled in membranesand matrices by varying the amount of p-GlcNAc material used, and by theaddition of certain solvents such as methanol or ethanol, with ethanolbeing preferred, in specific amounts, ranging from about 5% to about40%, prior to the formation of membranes and/or matrices. In general,the greater the percentage of solvent, the smaller the average pore sizeformed will be. The Example presented, below, in Section 15,demonstrates the synthesis and characterization of such porous p-GlcNAcstructures.

[0188] Thus, the p-GlcNAc may be formulated for administration byinhalation or insufflation (either through the mouth or the nose) ororal, buccal, parenteral or rectal administration.

[0189] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate) Thep-GlcNAc may be used in place of, or in addition to, the excipients andfillers. Tablets may be coated using p-GlcNAc using methods well knownin the art. Liquid preparations for oral administration may take theform of, for example, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending acents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats) emulsifying agents (e., lecithin or acacia);non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

5.6. Uses

[0190] The p-GlcNAc of the invention, as well as its deacetylatedderivatives and their derivatizations, such as those described, above,in Section 5.4, and reformulations, such as those described above, inSection 5.5, have a variety of uses. For example, the non-toxic,non-pyrogenic, biodegradable, and biocompatible properties of themolecules of the invention, in addition to the advantageous propertiesof the p-GlcNAc and its derivatives, as described herein, lendthemselves to applications in such diverse fields as agriculture,cosmetics, the biomedical industry, animal nutrition and health, and thefood, chemical, photographic, and pharmaceutical industries.

5.6.1. Biomedical Uses of p-GlcNAc Materials 5.6.1.1. DrugImmobilization/Delivery Uses

[0191] Biomedical uses of p-GlcNAc material may include, for example,enzyme and/or drug immobilization/delivery methods. The p-GlcNAc/drugformulations of the invention can provide additional benefits to theknown drug formulations, including, for example, increasedeffectiveness, reduced toxicity and improved bioavailability. SuchpGlcNAc/drug formulations can act as therapeutic agents which mayinclude, but are not limited to, antitumor agents, antibiotics,antibacterials, antifungals, anti-virals and anti-inflammatory drugs.

[0192] The drug/p-GlcNAc compositions can be formulated, for example, byimmobilizing a given drug by covalent or non-covalent attachment to, thep-GlcNAc or p-GlcNAc derivates of the invention. Techniques for covalentor non-covalent attachment are well known to those skilled in the art,and may be as described. The drug/p-GlcNAc compositions of the inventioncan also be formulated, for example, by encapsulating a given moleculewithin p-GlcNAc or an p-GlcNAc derivative. Encapsulation methods aredescribed, above, in Section 5.4.

[0193] Upon attachment or encapsulation of the molecule, assays commonlyemployed by those of skill in the art may be utilized to test theactivity of the particular molecule or molecules attached, therebyensuring that an acceptable level of biological activity (e.g., atherapeutically effective activity) is retained by the attached moleculeor encapsulated molecule.

[0194] p-GlcNAc/drug formulations and p-GlcNAc/drug encapsulations maybe delivered to a patient via a variety of routes using standardprocedures well known to those of skill in the art. For example, suchdelivery may be site-specific, oral, nasal, intravenous, subcutaneous,intradermal, transdermal, intramuscular or intraperitonealadministration. Both p-GlcNAc/drug formulations and p-GlcNAc/drugencapsulations may be formulated to function as controlled, slow releasevehicles, as described in this Section, below.

[0195] With respect to site-specific delivery, administration methodsmay include, but are not limited to injection, implantation,arthroscopic, laparoscopic or similar means. p-GlcNAc membranes and/orgels as well as microspheres and sponges are preferred for suchsite-specific delivery methods.

[0196] There are numerous advantages in using the p-GlcNAc based drugdelivery systems of the invention. Traditional drug administration byinjection is commonly used with proteins and many other drugs. However,repeated doses lead to oscillating blood drug concentrations and affectpatient comfort and compliance. Oral administration can be advantageoussince it allows for a more varied load of the drug to be released and isless discomforting to the patient. However, proteins and other compoundsare denatured and degraded in the stomach.

[0197] An improved oral administration, however, is achieved by thecompound-containing p-GlcNAc molecules or p-GlcNAc compoundencapsulations of the invention by providing a protective environmentfor the drug once administered. For example, the p-GlcNAc of theinvention protects a compound, such as a protein, from the acidic andenzymatic environment of the stomach. The p-GlcNAc system releases thecompound via diffusion and/or encapsulation degradation once it reachesthe intestinal region, where it is effectively absorbed into the bloodstream. These p-GlcNAc systems of the invention can be used, forexample, to deliver proteins, such as, insulin, as well as many othercompounds. Liposomes coated with p-GlcNAc derivatives or p-GlcNAcderivatives-alginate encapsulations are preferred for such oral deliverymethods.

[0198] Upon introduction of the compound-containing p-GlcNAc and/orp-GlcNAc encapsulations into a patient, the p-GlcNAc of the inventionbiodegrades, such that the attached compounds are gradually releasedinto the bloodstream of the patient, thus providing a method forcontrolled compound or drug delivery.

[0199] Deacetylated or partially deacetylated p-GlcNAc species may beproduced having a predictable rate of biodegradability. For example, thepercentage of deacetylation affects the rate at which the p-GlcNAcspecies degrades. Generally, the higher the percentage of deacetylation,the faster the rate of biodegradability and resorption will be. Thus,the degree of p-GlcNAc biodegradability and the in vivo rate ofresorption may be controlled during the p-GlcNAc's production. Examplesof the production and characterization of such p-GlcNAc materials arepresented in Section 18, below. p-GlcNAc materials having suchcontrollable biodegradability rates may be formulated into membranes,gels, sponges, microspheres, fibers, and the like. These p-GlcNAcproducts adhere and mold to tissues, both soft and hard tissues, in thehuman body with no need for suturing. The p-GlcNAc materials may, forexample, be applied during general or minimally invasive surgery, suchas laparoscopic surgery.

[0200] Compound-p-GlcNAc and p-GlcNAc encapsulations have a variety ofapplications as therapeutic drug delivery systems. Compounds which maybe encapsulated within or attached to (covalently or non-covalently) thep-GlcNAc, or a derivative of the invention are, for example, antitumorcompounds, antibiotics, antibacterials, antifungals, antivirals, smallpeptide and non-peptide molecules, vitamins and other health-relatedfood additives. Anti-tumor drugs which can be attached to, orencapsulated within the p-GlcNAc of the invention include, but are notlimited to, those listed in this Section, below.

[0201] Additionally, combinations of two or more molecules may beencapsulated within or attached to the p-GlcNAc of the invention toprovide a synergistic effect. For example, anti-tumor agents such asthioguanine combined with cytosine arabinoside (ara-C) are contemplatedfor use in the invention as an improved treatment for acutenonlymphocytic leukemia. Other synergistic combinations includetamoxifen with cisplatin for breast cancer, and prostaglandins withcisplatin for breast and prostate cancer. Additionally, many othersynergistic combinations of anti-cancer drugs, known to those of skillin the art, may be used with the p-GlcNAc and p-GlcNAc derivativeformulations of the invention.

[0202] Further, gene therapy agents, such as anti-sense DNA and ribozymesystems may also be encapsulated within the p-GlcNAc or p-GlcNAcderivatives of the invention. Immunotherapeutic compounds (e.g.,vaccines), such as tumor specific antigens, that can elicit an immuneresponse (e.g., a cytotoxic T-lymphocyte response) against a specifictumor type (e.g., melanoma) can also be attached to, or encapsulatedwithin the p-GlcNAc of p-GlcNAc derivatives of the invention by methodsknown in the art.

[0203] The drug delivery systems described herein are feasible for usewith any anti-tumor drug. For example, the use of such anti-tumor drugdelivery systems is demonstrated in the Example presented in Section 20,below. Such drugs are well known to those of skill in the art, and maybe formulated into p-GlcNAc gels or membranes, for example, so as toprovide site-specific slow-release delivery directly to the tumor or tothe region vacated by the tumor following surgery. Such an immobilizedslow-release p-GlcNAc drug product can act as an important initialdefensive procedure after surgery. Such p-GlcNAc anti-tumor drugdelivery systems are particularly useful in treating tumors which aretotally or partially inaccessible through surgery, such as, for example,is the case with certain brain tumors.

[0204] Additionally, the use of p-GlcNAc/compound and p-GlcNAcencapsulations of the invention for the development of new anti-tumordrug formulations is desirable given that the p-GlcNAc polymer haschemical properties and characteristics making possible the formulationand delivery of some drugs that have heretofore been difficult toformulate and deliver. For example, taxol, a microtubule spindleinhibitor drug used to treat breast cancer, is hydrophobic and requiresthe addition of polyoxyethylated castor oil in order to solubilize it asa liquid infusion for intravenous delivery. The hydrophobic nature oftaxol makes it an ideal compound for formulation with p-GlcNAc polymermaterials for topical controlled release delivery. The Example presentedin Section 23, below, presents such a p-GlcNAc/taxol formulation.Additional targets for p-GlcNAc anti-tumor systems include, but are notlimited to, skin, GI tract, pancreatic, lung, breast, urinary tract anduterine tumors, and HIV-related Kaposi's sarcomas.

[0205] For example, anti-tumor drugs that may be formulated with thep-GlcNAc and p-GlcNAc encapsulation system of the invention include, butare not limited to, the following categories and specific compoundsalkylating agents, antimetabolite agents, anti-tumor antibiotics, vineaalkaloid and epidophyllotoxin agents, nitrosoureas, enzymes, synthetics,hormonal therapeutic biologics and investigational drugs.

[0206] Such alkylating agents may include, but are not limited tonitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan,thiptepa and busulfan.

[0207] Antimetabolites can include, but are not limited to,methotrexate, 5-fluorouracil, cytosine arabinoside (ara-C),5-azacytidine, 6-mercaptopurine, 6-thioguanine, and fludarabinephosphate. Antitumor antibiotics may include but are not limited todoxorubicin, daunorubicin, dactinomycin, bleomycin, mitomycin C,plicamycin, idarubicin, and mitoxantrone. Vinca alkaloids andepipodophyllotoxins may include, but are not limited to vincristine,vinblastine, vindesine, etoposide, and teniposide.

[0208] Nitrosoureas such as, but not limited to carmustine, lomustine,semustine and streptozocin. Enzymes can include, but are not limited toL-asparagine.

[0209] Synthetics can include, but are not limited to Dacrabazine,hexamethylmelamine, hydroxyurea, mitotane procabazine, cisplatin andcarboplatin.

[0210] Hormonal therapeutics can include, but are not limited tocorticosteriods (cortisone acetate, hydrocortisone, prednisone,prednisolone, methyl prednisolone and dexamethasone), estrogens,(diethylstibesterol, estradiol, esterified estrogens, conjugatedestrogen, chlorotiasnene), progestins (medroxyprogesterone acetate,hydroxy progesterone caproate, megestrol acetate), antiestrogens(tamoxifen), aromastase inhibitors (aminoglutethimide), androgens(testosterone propionate, methyltestosterone, fluoxymesterone,testolactone), antiandrogens (flutamide), LHRH analogues (leuprolideacetate), and endocrines for prostate cancer (ketoconazole).

[0211] Biologics can include, but are not limited to interferons,interleukins, tumor necrosis factor, and biological response modifiers.

[0212] Investigational Drugs can include, but are not limited toalkylating agents such as Nimustine AZQ, BZQ, cyclodisone, DADAG,CB10-227, CY233, DABIS maleate, EDMN, Fotemustine, Hepsulfam,Hexamethylmelarine, Mafosamide, MDMS, PCNU, Spiromustine, TA-077, TCNUand Temozolomide; antimetabolites, such as acivicin, Azacytidine,5-aza-deoxycytidine, A-TDA, Benzylidene glucose, Carbetimer, CB3717,Deazaguanine mesylate, DODOX, Doxifluridine, DUP-785, 10-EDAM,Fazarabine, Fludarabine, MZPES, MMPR, PALA, PLAC, TCAR, TMQ, TNC-P andPiritrexim; antitumor antibodies, such as AMPAS, BWA770U, BWA773U,BWA502U, Amonafide, m-AMSA, CI-921, Datelliptium, Mitonafide,Piroxantrone, Aclarubicin, Cytorhodin, Epirubicin, esorubicin,Idarubicin, Iodo-doxorubicin, Marcellomycin, Menaril, Morpholinoanthracyclines, Pirarubicin, and SM-5887; microtubule spindleinhibitors, such as Amphethinile, Navelbine, and Taxol; thealkyl-lysophospholipids, such as BM41-440, ET-18-OCH₃, andHexacyclophosphocholine; metallic compounds, such as Gallium Nitrate,CL286558, CL287110, Cycloplatam, DWA2114R, NK121, Iproplatin,Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium compounds; andnovel compounds such as, for example, Aphidoicolin glycinate, Ambazone,BSO, Caracemide, DSG, Didemnin, B, DMFO, Elsamicin, Espertatrucin,Flavone acetic acid, HMBA, HHT, ICRF-187, Iododeoxyuridine, Ipomeanol,Liblomycin, Lonidamine, LY186641, MAP, MTQ, Merabarone SK&F104864,Suramin, Tallysomycin, Teniposide, THU and WR2721; a nd Toremifene,Trilosane, and zindoxifene.

[0213] Antitumor drugs that are radiation enhancers are preferred forinstances in which radiation therapy treatment is to be prescribed,either in lieu of, or following surgery. Examples of such drugs include,for example, the chemotherapeutic agents 5′-fluorouracil, mitomycin,cis-platin and its derivatives, taxol, doxorubicin, actinomycin,bleomycins, daunomycins, and methamycins.

[0214] Dose ranges for anti-tumor drugs may be lower than, equal to orgreater than the typical daily doses prescribed for systemic treatmentof patients. Higher doses may be tolerated in that the drugs aredelivered locally at the site of a tumor, whereas other tissues,therefore, including blood cells, are not as readily exposed to thedrugs. For example, dosages of 5′-FU equivalent to 50% of the standarddosages used to treat colorectal cancer with 5′-FU in humans (300-450mg/m² i.v. daily for 5 days) resulted in an 80-90% reduction in volumeof ectopic HT29 colon cancer tumor implants in scid mice. The use of thep-GlcNAc membrane as a drug delivery matrix for the administration of5′-FU reduced the dosage required to dramatically reduce tumor volume by50% as compared to intravenuous control animals. Details regarding thisdata can be found in Example 21, below.

[0215] Further, doses of such drugs are well known to those of skill inthe art and can be easily found in such compendia as the PHYSICIANS DESKREFERENCE, Medical Economics Data Publishers; REMINGTON'S PHARMACEUTICALSCIENCES, Mack Publishing Co.; GOODMAN & GILMAN, THE PHARMACOLOGICALBASIS OF THERAPEUTICS, McGraw Hill Publishers, THE CHEMOTHERAPY SOURCEBOOK, Williams and Wilkens Publishers, online services such as theCancer Lit®, U.S. National Cancer Institute database, as well as reportsof pharmacological studies such as “A MultiCenter Randomized Trial ofTrial of Two Doses of Taxol” Nabholtz, J. M., Gelmon, K., Bontenbal, M.et al. Medical Education Services Monograph—1994 Bristol-Myers SquibbCompany Publication; “Randomized Trial of Two Doses of Taxol inMetastatic Breast Cancer: An Interim Analysis” Nabholtz, J. M., Gelmon,K., Bontenbal, M., et al. 1993, Proc. Am. Clin. Oncol., 12:60. Abstract42 Alternatively, such doses can be routinely determined by usingstandard techniques well known to those of skill in the art such as, forexample, those described, below, at the end of this Section.

[0216] Certain anti-tumor agents are vesicants, including dactinomycin,daunomycin, doxorubicin, estramustine, mechlorethamine, mitomycin C,vinblastine, vincristine and videsine; while certain anti-tumor drugsare irritants, including carmustine, decarbazine, etoposide, mithrmycin,streptozocin and teniposide. Vesicants and irritants cause adverseside-effects including extravasation and irritation of tissues withpain, redness, swelling, and other symptoms. Further, tissue necrosiscan result from some of the side effects. The p-GlcNAc membrane and gelmaterials of the invention used for the topical, controlled release ofanti-tumor drugs have wound healing properties. Normal tissues that arein contact with vesicant or irritant anti-tumor drugs delivered by thep-GlcNAc membrane and gel formulations of the invention are, therefore,not as readily damaged and will heal faster due to the active healingeffects of the p-GlcNAc component of the anti-tumor drug-containingp-GlcNAc and p-GlcNAc-drug encapsulations of the invention.

[0217] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulations of the invention may, additionally, be used for thetreatment of infections. For such an application, antibiotics, eitherwater soluble or water insoluble, may be immobilized/formulated inp-GlcNAc based materials, such as, for example, gels and membranes.Antibiotics are well known to those of skill in the art, and include,for example, penicillins, cephalosporins, tetracyclines, ampicillin,aureothicin, bacitracin, chloramphenicol, cycloserine, erythromycin,gentamicin, gramacidins, kanamycins, neomycins, streptomycins,tobramycin, and vancomycin Doses of such drugs are well known to thoseof skill in the art, and may, alternatively, routinely be determinedusing standard techniques well known to those of skill in the art, suchas, for example, are described, below, at the end of this Section.

[0218] Such p-GlcNAc antibiotic products may be used to treat bacterialinfections that occur either externally, e.g., on skin, scalp, dermalulcers or eyes, or internally, e.g., localized infections of the brain,muscles, abdomen. A prominent application is for treatment ofHIV-related opportunistic infections.

[0219] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulation systems of the invention may be formulated withanti-inflammatory drugs to control dysfunctional activity of theinflammatory and immune processes. For example, p-GlcNAc may beformulated with non-steroidal anti-inflammatory drugs (NSAIDs) and usedto the reduction of local pain and inflammation induced by diseases suchas rheumatoid arthritis, osteoarthritis and systemic lupus, to name afew. The localized delivery of such NSAIDs using the p-GlcNAc gel ormembrane/drug delivery systems of the invention may serve to reduceNSAID side effects, which may include gastric irritation, azotemia,platelet disfunction and liver function abnormalities. NSAIDs are wellknown to those of skill in the art and include inhibitors ofcycloxygenase, such as aspirin, etodolac, fenoprofen and naproxen. Otheranti-inflammatory drugs may be utilized as part of thecompound-containing p-GlcNAc and p-GlcNAc encapsulation systems of theinvention, such as, for example, inhibitors of lipid inflammatorymediators, such as leucotrienes. Doses for such drugs are well known tothose of skill in the art, and may, alternatively, routinely bedetermined using standard techniques well known to those of skill in theart, such as, for example, are described, below, at the end of thisSection.

[0220] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulation systems of the invention may additionally be formulatedwith antifungal agents, using techniques described above, for thetreatment of specific fungal diseases. Antifungal agents are well knownto those of skill in the art, and may include, for example,amphotericin, anisomycin, antifungone, blastomycin, griseofulvins, andnystatin. Doses of such drugs are well known to those of skill in theart, and may, alternatively, routinely be determined using standardtechniques well known to those of skill in the art, such as, forexample, are described, below, at the end of this Section.

[0221] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulation systems of the invention may also be formulated withantiprotozoal agents, using techniques described above, for thetreatment of specific protozoal infections. Antiprotozoal agents arewell known to those of skill in the art, and may include, for example,antiamoebin, antiprotozin, monomycin, paromomycin and trichomycin. Dosesof such drugs are well known to those of skill in the art, and may,alternatively, routinely be determined using standard techniques wellknown to those of skill in the art, such as, for example, are described,below, at the end of this Section.

[0222] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulation systems of the invention may be formulated withspermicidal compounds, using techniques such as those described, above,to produce effective contraceptives. Appropriate spermicides are wellknown to those of skill in the art. Doses of such spermicides are wellknown to those of skill in the art, and may, alternatively, routinely bedetermined using standard techniques well known to those of skill in theart, such as, for example, are described, below, at the end of thisSection.

[0223] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulation systems of the invention may, still further, be formulatedusing therapeutic protein agents. Such formulations may be producedusing, for example, techniques such as those described above. Byutilizing such p-GlcNAc therapeutic protein systems, it is possible todeliver specific proteins directly to desired target sites and to effectslow release of the proteins at such sites. Examples of possibleproteins include, but are not limited to insulin, monoclonal antibodies,breast cancer immunotoxin, tumor necrosis factor, interferons, humangrowth hormone, lymphokines, colony stimulating factor, interleukins andhuman serum albumin. Doses of such therapeutic protein agents are wellknown to those of skill in the art and may be found in pharmaceuticalcompedia such as the PHYSICIANS DESK REFERENCE, Medical Economics DataPublishers; REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co.;GOODMAN & GILMAN, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw HillPubl., THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers,and may, alteratively, routinely be determined using standard techniqueswell known to those of skill in the art, such as, for example, aredescribed, below, at the end of this Section.

[0224] The compound-containing p-GlcNAc and p-GlcNAc-compoundencapsulation delivery systems of the invention can also be formulatedas nutritional and vitamin supplements.

[0225] Because the p-GlcNAc materials of the invention are themselvesimmunoneutral, in that they do not elicit an immune response in humans,such p-GlcNAc devices, as described above, comprising p-GlcNAcmembranes, 3D porous matrices and/or gels that harbor immobilized drugs,may deliver such drugs in a manner that there is no immune response.Certain additional materials, such as natural alginates and syntheticpolymers, may be used in some cases to construct such devices incombination with the p-GlcNAc material. For instance, a polymericdelayed-release drug delivery system can be manufactured in a mannersimilar to that suggested by A. Polk (Polk, A. et al., 1994, J. ofPharmaceutical Sciences, 83(2):178-185). In such a procedure,deacetylated p-GlcNAc is reacted with sodium alginate in the presence ofcalcium chloride to form microcapsules containing the drug to bedelivered and released under appropriate conditions and over a certainlapse of time.

[0226] The therapeutically effective doses of any of the drugs or agentsdescribed above, in conjunction with the p-GlcNAc-based systemsdescribed herein, may routinely be determined using techniques wellknown to those of skill in the art. A “therapeutically effective” doserefers to that amount of the compound sufficient to result inamelioration of symptoms of the processes and/or diseases describedherein. Toxicity and therapeutic efficacy of the drugs can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

[0227] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5.6.1.2. p-GlcNAc Cell Encapsulation Uses

[0228] p-GlcNAc encapsulated cells may be formulated, and such p-GlcNAcencapsulated cells may be administered to a patient, via standardtechniques well known to those of skill in the art. See, for example,the techniques described, above, in Section 5.6.1.1. Alternatively, see,for example, Aebisher et al. (Aebisher, P. et al., in “Fundamentals ofAnimal Cell Encapsulation and Immobilization”, 1993, CRC Press, pp.197-224), Yoshioke et al., (Yoshioke, T. et al., 1990, Biotechnol.Bioeng. 35:66) and U.S. Pat. No. 4,749,620, each of which isincorporated herein by reference in its entirety. Cells may beencapsulated by, on, or within p-GlcNAc or partially deacetylatedp-GlcNAc membranes, three-dimensional p-GlcNAc porous matrices, orp-GlcNAc gels.

[0229] Three-dimensional matrices can be seeded with cells and used incertain applications without further encapsulation. Alternatively, cellscan be encapsulated into microspheres or droplets of p-GlcNAc-basedpolymer gels such as, for example, a p-GlcNAc-lactate polyelectrolytepolymer (a polycationic polymer). Gels, droplets or microspheres intowhich cells have been encapsulated may then be coated with a secondpolyelectrolyte of opposite charge (e.g., with a polyanion, such as analginate) to form an outer capsule which provides immuno-isolation forthe encapsulated cells, thus reducing the risk of immune rejection bythe host organism.

[0230] Additionally, cells entrapped in p-GlcNAc gels, three-dimensionalp-GlcNAc matrices, or both, can be loaded into thermoplastic capsules inyet another method of formulation. Thermoplastic-based capsules can alsobe utilized to provide immuno-protection for implanted cells in a hostorganism. Such thermoplastic capsules are made of materials such ashydroxyethyl methylacrylate-methylmethacrylate copolymer (HEMA-MMA).Thermoplastic-derived microcapsules are formed, for example, by thecoextrusion of a solution of HEMA-MMA in polyethylene glycol and thecell-containing p-GlcNAc matrix and/or gel medium, into an appropriateorganic solvent such as hexadecane. See, for example, the methoddescribed by Aebisher et al. (Aebisher, P. et al., in “Fundamentals ofAnimal Cell Encapsulation and Immobilization”, 1993, CRC Press, pp.197-224).

[0231] The p-GlcNAc cell encapsulations have a variety of applications.First, they may be utilized for the delivery of therapeutic compounds,synthesized and secreted by cells attached to and encapsulated in themembranes, matrices or gels.

[0232] For example, and not by way of limitation, the p-GlcNAc/cellencapsulations may be used for delivery of insulin in the treatment ofdiabetes, nerve growth factor for the treatment of Alzheimer's disease,factor VIII and other clotting factors for the treatment of hemophilia,dopamine or the treatment of Parkinson's disease, enkephalins viaadrenal chromaffin cells for the treatment of chronic pain, dystrophinfor the treatment of muscular dystrophy, and human growth hormone forthe treatment of abnormal growth. For example, dopamine-producingadrenal chromaffin cells may be encapsulated for the treatment ofParkinson's Disease and for the relief of chronic pain. Also, pancreaticislet cells may be encapsulated for the treatment of insulin-dependentdiabetes mellitus. Further, the p-GlcNAc/cell encapsulations may be usedfor delivery of the above peptides and proteins, or other peptides andproteins, as gene products in vivo for use in gene therapies. Forexample, using recombinant DNA techniques, a gene for which a patient isdeficient could be placed under the control of viral or tissue specificpromotor. The recombinant DNA construct containing the gene could beused to transform or transfect a host cell which is cloned and thenclonally expanded and is engineered so as to secrete the said geneproduct then encapsulated into the p-GlcNAc/cell encapsulations of theinvention and used to deliver the gene product as a therapy.Additionally, such encapsulated cells may act to compensate for the lossof a specific organ or tissue and/or may be used to augment the reducedfunction of such a specific tissue or organ of the body. For example,encapsulated liver cells may be used to augment reduced liver function,and may, for example, be utilized as a temporary measure prior to theadministration of a liver transplant. As another example, encapsulatedthyroid, parathyroid or pancreas cells may be administered to compensatefor the loss of these glands.

[0233] Because the p-GlcNAc materials of the invention are themselvesimmunoneutral, as they do not elicit an immune response in humans, it ispossible to engineer and construct devices consisting of p-GlcNAcmembranes, three-dimensional porous p-GlcNAc matrices and/or p-GlcNAcgels that harbor attached cells which can deliver cell-basedtherapeutics in a manner such that the cells are immuno-isolated, i.e.,no anti-cell host immune response is elicited. Certain additionalmaterials, such as, for example, natural alginates and syntheticpolymers, may be used to construct such devices in addition to thep-GlcNAc material itself.

[0234] p-GlcNAc/cell encapsulation compositions may additionally beutilized for the delivery of cells to seed tissue regeneration.Applications of specific cell types encapsulated for the seeding of cellgrowth leading to tissue regeneration at the site of an injury mayinclude, but are not limited to regeneration of skin, cartilage, nerves,bone, tendon, ligaments, liver and blood vessels. The tissueregeneration applications of cells encapsulated in p-GlcNAc materialsare advantageous, in part, because of the ability of the p-GlcNAcmaterial to adhere to injured tissue, to provide a substrate formammalian cell growth, and to undergo bioresorption coincident with thegrowth of new healthy tissue during the tissue regeneration process atthe site of injury.

5.6.1.3. Utilizing p-GlcNAc Materials for the Prevention ofPost-Surgical Adhesions

[0235] Additionally, p-GlcNAc membranes may be used to provide abiodegradable, biocompatible mechanical barrier to prevent post-surgicaladhesions. The Example presented in Section 17, below, demonstrate sucha p-GlcNAc application. Solid p-GlcNAc or p-GlcNAc derivativesformulated into membranes or sponges may be utilized for such anapplication. Preferred membranes are thin, generally less than about 1mm in thickness. Preferable p-GlcNAc derivatives are p-GlcNAcderivatives which have been about 50-80% deacetylated. Such p-GlcNAcderivatives will generally be resorbed approximately 7-21 dayspost-implantation.

[0236] Liquid p-GlcNAc derivatives are also suitable for use in theprevention of post-surgical adhesions. Preferable liquid p-GlcNAcderivatives for such an application are deacetylated p-GlcNAc saltderivatives and carboxymethyl p-GlcNAc derivatives. A p-GlcNAcderivative which is particularly preferred for the prevention ofpost-surgical adhesions is a p-GlcNAc-lactate derivative, especially ap-GlcNAc-lactate gel derivative. As used herein, the termp-GlcNAc-lactate means that the lactic acid moiety is functionallyattached to a partially or fully deacetylatedpoly-β-1→4-N-acetylglucosamine of the invention. Such p-GlcNAc-lactatederivatives may be formulated using propylene glycol and water, as, forexample, described in Section 17.1. p-GlcNAc-lactate derivatives may beproduced having high and low viscosities, which allows for the abilityto tailor the p-GlcNAc used to the specific indication of interest. Forexample, it may be useful to use a p-GlcNAc product having a lowerviscosity for delivery through a syringe or via a spray, while it may bedesirable to use a p-GlcNAc product having a higher viscosity, andtherefore greater lubrication properties, when the indication is anorthopedic one.

[0237] For the prevention of post-surgical adhesions, solid p-GlcNAcformulations are suitable for clearly circumscribed wound sites. Suchp-GlcNAc formulations should be applied following the surgical procedureand the material should completely cover the traumatized tissue. It canbe applied either in conjunction with either general or minimallyinvasive (e.g., laparoscopic) surgical procedures. The solid p-GlcNAcformulations can be cut and applied using standard surgical proceduresand instrumentation well known to those of skill in the art.

[0238] The liquid p-GlcNAc formulations can be applied, for theprevention of post-surgical adhesions, in larger areas prone to formsuch postoperative adhesions. The p-GlcNAc-lactate gel, for example, canbe applied before the surgical procedure to provide additionallubrication and thus reduce the amount of traumatized tissue.Alternatively, the liquid p-GlcNAc formulation, such asp-GlcNAc-lactate, can be applied following the surgical procedure toform a physical barrier to prevent postoperative adhesion formation.

[0239] The p-GlcNAc material can be painted, sprayed or dropped from asyringe device onto the wounded site. In laparoscopic procedures, lowviscosity materials can, for example, be delivered with standard suctionirrigation devices. Higher viscosity materials will require pressure toreach its target. The pressure can be provided by a compressed gaspowered piston or a syringe type device.

[0240] The amount of liquid p-GlcNAc formulation, such as thep-GlcNAc-lactate gel formulation, required for prevention ofpost-surgical adhesions is proportional to the extent of the traumatizedtissue. The p-GlcNAc material administered should be applied in therange of 0.1 ml to 1.5 ml per sq. cm of surface area.

[0241] Various animal models which are known in the art can be used totest the post-surgical adhesion formulations of the invention. Theseinclude, but are not limited to, the Rat cecum model (Harris, E. W., etal., 1992, Journal of Investigative Surgery, 5:260; and the Rabbituterine horn model (Diamond, M. P., et al., 1987, Microsurgery, 8:197.

5.6.1.4. Biodegradable p-GlcNAc Barriers

[0242] p-GlcNAc materials having a controllable rate of biodegradationmay be useful as, for example barriers having a variety of applications.For example, such barriers may be utilized to promote hemostasis. Thesuccessful use of such a hemostatic p-GlcNAc application is demonstratedin the Example presented, in Section 19, below. Additionally,p-GlcNAc-based material, such as thick gels, sponges, films andmembranes may be used for such hemostatic applications. The p-GlcNAcbased materials, when applied directly to bleeding surfaces, arrestbleeding by providing a mechanical matrix that promotes clotting. Thep-GlcNAc based materials adhere to the site of application and seal theboundaries of the wound. This reduces the amount of blood loss, protectsthe forming clot and facilitates the clotting process.

[0243] The preferred solid materials for such an application aredeacetylated p-GlcNAc membranes and sponges. The preferred thick gelscan be formulated from water plus soluble derivatives like deacetylatedp-GlcNAc salts and carboxymethyl p-GlcNAc derivatives. The thick gels ofthe invention should have a viscosity of 50,000 cps or greater. Forexample, and not by way of limitation, one formulation of such a gel ispresented in Example 17.1 in formulating p-GlcNAc-lactate as a gel.

[0244] Applications for the hemostatic agents described above include,but are not limited to, uses in diagnostic procedures such as biopsywounds in, for example, liver and kidney; in trauma wounds, for example,spleen, liver and blood vessel injuries; in standard and minimallyinvasive surgical procedures, for example, endometriosis surgery andoperations on the gallbladder; in soft and hard tissue wound repair, forexample, skin wounds and burn healing; in surgical procedures, inparticular, for splenic wounds; and for blood vessel puncture diagnosticand treatment procedures such as catheterization and balloon angioplastyprocedures.

[0245] The solid p-GlcNAc based materials of the invention can beapplied using standard surgical procedures, and can be used with bothstandard and minimally invasive surgical interventions. The thick gelsof the invention can be delivered, for example, by extrusion from asyringe type device or in combination with a membrane or film. Themembrane or film can be manufactured from a p-GlcNAc based material orother natural or synthetic materials.

[0246] In connection with the blood vessel puncture procedures mentionedabove, the hemostatic agents of the invention may be applied at the timewhen a catheter sheath is being removed from a blood vessel. A devicethat detects the removal of the catheter sheath from the blood vesselcan be developed using electronic or mechanical systems that monitorchemical, physical or other differences between the tissue inside andoutside of the vessel. For example, the differential in fluid dynamicsor heat dissipation can be detected when a probe is removed from thevessel; at that point a signal is sent to initiate the application ofthe hemostatic agent.

[0247] Further, p-GlcNAc materials may be utilized to provideperiodontal barriers for the separation of soft and hard tissue duringthe repair process following periodontal surgery in order to promoteuniform tissue repair, to provide biodegradable contact lenses, cornealshields or bone grafts, to provide surgical space fillers, to promotesoft tissue augmentation, particularly in the skin for the purpose ofreducing skin wrinkles, and as urinary sphincter augmentation, for thepurpose of controlling incontinence.

5.6.1.5. Other Biomedical Uses of p-GlcNAc Materials

[0248] Other biomedical uses of p-GlcNAc materials include, for example,the use of such materials as cell culture substrates. For example, asshown in the Working Example presented in Section 12, below, thep-GlcNAc of the invention acts as a very efficient substrate formammalian cells grown in culture. Further, three-dimensionalconfigurations of p-GlcNAc may be used as medium components which willallow three-dimensional cell culture growth.

[0249] The cell substrate capabilities of the p-GlcNAc of the inventionmay also be utilized in vivo. Here, the p-GlcNAc of the invention, or aderivative thereof, as described herein, may act to facilitate tissueregeneration (e.g., regeneration of connective tissue covering teethnear the gum line, vascular grafts, ligament, tendon, cartilage, bone,skin, nerve tissues). The p-GlcNAc molecules of the invention may,therefore, for example, have extensive plastic surgery applications.

[0250] Deacetylated p-GlcNAc is preferred for use as a sealant ofvascular grafts. Deacetylated p-GlcNAc derivatives such asN-carboxymethyl and N-carboxybutyl deacetylated p-GlcNAc are preferredas tissue regeneration reagents. N-carboxymethyl deacetylated p-GlcNAcmay, for example, be inoculated into the cornea to induceneovascularization.

[0251] Further biomedical applications of the p-GlcNAc of the inventionor of its derivatives, as described herein, may involve the molecules'use in wound dressing, wound healing ointments, and surgical sutures,sponges, and the like. Such promotion of wound healing and reduction ofscarring is illustrated, below, in examples 17 and 22. These propertiesare useful in stimulating tissue repair, and accelerates, strengthen andimprove the quality of healing. p-GlcNAc based materials may be used forinjury related or surgically induced wounds in both soft and hardtissue. For example, such wounds include venous stasis ulcers, burnhealing and surgical wounds in the eye or other tissues where quality ofhealing or fibrosis may be important.

[0252] Still further, such molecules may be used, for example, in thetreatment of osteoarthritis, in the reduction of blood serum cholesterollevels, as anti-viral agents, as anti-bacterial agents, asimmunomodulators, as anticoagulants, as dialysis and ultrafiltrationmembranes, as anti-tumor agents, as contact lens material, and as oraladsorbents for uremic toxins when administered to kidney failurepatients. Microcrystalline p-GlcNAc suspensions or water solublep-GlcNAc derivatives are preferred for the treatment of arthritis, by,for example, injection directly into arthritic joints.

[0253] p-GlcNAc has additional applications as a component of artificialor donor skin. For example, p-GlcNAc, preferably as non-woven p-GlcNAcfilms, may be applied to split thickness skin donor sites, over, forexample, donor dermis.

[0254] Deacetylated p-GlcNAc to which a protease, such as pepsin, hasbeen attached may be used for the controlled digestion of proteins incontact with such p-GlcNAc/protease compounds.

5.6.2. Agricultural Uses of p-GlcNAc Materials

[0255] The p-GlcNAc of the invention or its derivatives may be used invarious agricultural applications, as well. Such applications include,but are not limited to insecticide, fungicide, bactericide, andnematocide applications. N-carboxymethyl deacetylated p-GlcNAcderivatives are preferred for use as effective bacteriostatic reagents.N-alkyl p-GlcNAc derivatives may be preferred for fungicideapplications. Additionally, the molecules of the invention may be usedin various soil treatment applications, including, but not limited to,fertilizer compositions. Further, controlled release of agrochemicalsmay be achieved by entrapping such chemicals via the immobilization,encapsulation, and other methods described, above, in this Section.Additionally, analogs of, for example, Rhizobium nodulation factorsand/or nitrogen fixation inducers may be immobilized onto, andadministered via, the p-GlcNAc and/or p-GlcNAc derivatives of theinvention.

5.6.3. Nutrition/Food Industry Uses of p-GlcNAc Materials

[0256] The p-GlcNAc of the invention and its derivatives as describedherein additionally have applications in the fields of animal and humannutrition. For example, the molecules of, the invention may be used asfeed ingredients Techniques such as those described, above, in thisSection, may be used in the production of controlled release products inanimal systems. Additionally, the biomedical applications describedabove may be utilized in animal systems by incorporating routinemodifications well known to those of ordinary skill in the art.

[0257] Food industry applications of the p-GlcNAc of the invention andof its derivatives, as described herein, may include, but are notlimited to anticholesterol (i.e., hypocholesterolemic compounds),fat-binding compounds, emulsifiers, carriers, preservatives, seasonings,and food texturizers, in addition to fruit coatings, and food packagingproducts. For example, in terms of cholesterol and fat binding, p-GlcNAcderivatives exhibit strong binding activity towards lipids. Deacetylatedand appropriately hydrolyzed p-GlcNAc derivatives also bind to lipidsand dietary cholesterol. (Vahouny, G. V., 1983, Journal of ClinicalNutrition 38:278-284). It is possible that p-GlcNAc exerts ahypocholestrolemic effect by forming micelles with cholesterol andlipids, causing them eventually to be excreted (Hirano, S., 1990, in,“The International Symposium on Chitin Derivatives in Life Sciences”).

5.6.4. Cosmetic Uses of p-GlcNAc Materials

[0258] Cosmetic applications of the p-GlcNAc of the invention mayinclude, but are not limited to, the production of products for hair andskin care. Skin care products may include, for example, cosmeticsutilizing deacetylated p-GlcNAc salts, carboxymethyl p-GlcNAc-containingproducts, and cosmetic packs containing deacetylated p-GlcNAc and suchderivatives as hydroxypropyl-, N-succinyl-, and quaternary p-GlcNAcderivatives. Hair products may include, for example, carboxymethylp-GlcNAc-containing products, and film-forming p-GlcNAc derivatives.

5.6.5. Chemical Engineering Applications of p-GlcNAc Materials

[0259] The p-GlcNAc of the invention and its derivatives have a varietyof applications that are useful in the chemical engineering industry.For example, p-GlcNAc may be used as a coupling agent for adhesion ofmetals to polymers, membranes formed by glycol p-GlcNAc may be used indesalination applications, and membranes formed by other p-GlcNAcderivatives may be used for transport of halogen ions. Otherapplications may include the production of flame retardants, and themanufacture of metal chelating compounds and compounds capable ofremoving trace and heavy metals from liquids as well as water-solubleindustrial pollutants, such as PCBs, for example. p-GlcNAc and/orp-GlcNAc derivatives may be used in photographic applications. Forexample, the ability of p-GlcNAc and/or p-GlcNAc derivatives to chelatemetals, such as silver halides, may be utilized by contactingphotographic solutions to recast mats, such as thin membranes, ofp-GlcNAc and/or p-GlcNAc derivatives.

6. EXAMPLE: PHYSICAL CHARACTERIZATION OF PREPARATIONS OF PURE p-GlcNAC

[0260] Presented in this Example, are circular dichroism (CD) andinfra-red spectra (IR) analyses of p-GlcNAC and deacetylated p-GlcNACmembranes.

6.1. Materials and Methods

[0261] p-GlcNAC and Commercial “Chitin” Preparations:

[0262] The p-GlcNAc used in the CD studies was prepared using theMechanical Force purification method described, above, in Section 5.3.1.

[0263] Commercial “chitin” was purchased from NovaChem, Ltd., PO Box1030 Armdale, Halifax, Nova Scotia, Canada, B3L 4K9.

[0264] The p-GlcNAC membranes used in the IR studies were prepared byeither the Mechanical Force purification method as described, above, inSection 5.3.1, or by the Chemical/Biological purification method, asdescribed, above, in Section 5.3.2, as indicated.

[0265] The commercial “p-GlcNAc” preparations were cast into membranesby dissolving in a dimethylacetamide solution containing 5% lithiumchloride, and layering onto distilled, deionized water until membranesprecipitated.

[0266] p-GlcNAC derivatives and treatments: The Deacetylated p-GlcNACused in both the CD and IR studies was prepared by treatment of thep-GlcNAC with 50% NaOH at 60° C. for 2 hours. The heat-denaturedp-GlcNAC membranes used in the IR studies were modified by boiling in0.2 mM EDTA for 3 minutes. p-GlcNAc was autoclaved for 30 minutes at122° C.

[0267] CD techniques: Solid state CD techniques were carried outessentially according to Domard (Domard, A., 1986, Int. J. Macromol.8:243-246).

6.2. Results 6.2.1. CD Analysis

[0268] In the CD spectra obtained from untreated p-GlcNAc (FIG. 3A), theexpected n-π^(*) and π-π*^(*) optically active electronic transitions(220-185 nM) were observed due to the presence of the carbonyl group inthe acetyl moiety of p-GlcNAc. Such peaks are completely absent in theCD spectrum obtained from the deacetylated p-GlcNAc product, as shown inFIG. 3B.

6.2.2. IR Spectra Analysis

[0269] The IR spectra obtained in this study are consistent with thechemical structure of p-GlcNAc. Additionally, the sharp definition ofeach IR peak is indicative of the presence of an ordered and regular(i.e., pseudocrystalline) structure in the p-GlcNAc fibers. See FIG. 4Afor the IR spectrum of p-GlcNAc purified via the Mechanical Forcepurification method, and FIG. 4D for the IR spectrum of p-GlcNAcpurified via the Chemical/Biological method. For comparison, see FIG.4B, which demonstrates the IR spectrum of a commercial “chitin”preparation.

[0270] The IR spectrum obtained from the autoclaved p-GlcNAc material(FIG. 4E) does not differ visibly from the IR spectrum observed in FIG.4A. This data indicates that the p-GlcNAc material may be sterilized byautoclaving with no loss of polymer structure.

7. EXAMPLE: PURIFICATION OF p-GlcNAC USING THE MECHANICAL FORCEPURIFICATION METHOD

[0271] In this section, p-GlcNAC was purified using the Mechanical Forcetechnique described above, in Section 5.3.1.

7.1. Materials and Methods/Results

[0272] Diatom culture conditions: The diatom species Thalassiosirafluviatilis was grown in culture according the procedures described,above, in Sections 5.1 and 5.2.

[0273] SEM Procedures: The SEM techniques used here are as thosedescribed, below, in Section 12.1.

[0274] p-GlcNAc Purification procedure: p-GlcNAC was purified from thediatom culture by utilizing the Mechanical Force technique describedabove, in Section 5.3.1. Specifically, the p-GlcNAc fibers wereseparated from the diatom cell bodies by subjecting the contents of theculture to three short bursts of top speed mixing motion in a Waringblender. Total time of the three bursts was about one second. Theresulting suspension was centrifuged at 3500 rpm in a Sorvall GS-4 fixedangle rotor, for 20 minutes at about 10° C. The supernatant wasdecanted, and centrifuged again, this time at 4000 rpm, in a SorvallGS-4 fixed angle rotor for 20 minutes at about 10° C. Once again, thesupernatant was decanted and centrifuged at 4000 rpm at 10° C. The finalsupernatant of the third centrifugation was clear, with little, if any,visible flocs floating in the liquid. The clear supernatant was decantedinto a Buchner filtration unit equipped with a Supor-800 polyethersulfone filter membrane with 0.8 μm pore size (Gelman, Inc.), suctionwas then applied and the liquid was filtered from the fiber suspension,allowing the fibers to be collected on the membrane. The collectedfibers were washed with 1 liter of distilled, deionized H₂O at 70° C.When almost all of the water had been drained, fibers were washed, withsuction, with 1 liter of 1 N HCl at 70° C. When most of the acidsolution had been drained, the fibers were washed with 1 liter ofdistilled, deionized H₂O at 70° C., using suction. When most of the washwater had been drained, the fibers were washed with 1 liter of 95%ethanol at room temperature, and vacuum was applied. The filter membraneon which the white fiber membrane had been collected was then removedfrom the filtration unit and the membrane and its membrane support wasdried in a drying oven at 58° C. for 20 minutes, after which themembrane and its support was placed in a desiccator for 16 hours.

[0275] Following this purification procedure, the yield of p-GlcNAc froma 1000 ml culture was 6.85 milligrams per liter of diatom culture. SEMphotographs of the membrane formed by the collection of the p-GlcNACfibers via this technique is shown in FIG. 6.

8. EXAMPLE: PURIFICATION OF p-GlcNAC USING THE BIOLOGICAL/CHEMICALPURIFICATION METHOD

[0276] In this section, p-GlcNAC was purified using two of theChemical/Biological techniques described above, in Section 5.3.2.Briefly, p-GlcNAC was purified via HF treatment, in one case, and viaacid treatment/neutralization in the second case.

8.1. Materials and Methods/Results

[0277] Diatom culture conditions: The diatom species Thalassiosirafluviatilis was grown in a culture according to the proceduresdescribed, above, in Sections 5.1 and 5.2.

[0278] SEM procedures: The techniques utilized in this study were asdescribed, below, in Section 12.1.

[0279] Purification procedure: First, p-GlcNAC was purified by HFtreatment, the results of which are shown in FIG. 7. Specifically, undera fume hood, 2.42 ml of a 49% (29N) HF solution was added to the diatomcontents of the culture, at room temperature, for each 1000 ml of thevolume of the original cell culture, resulting in a 0.07 M HF solution.The mixture was then shaken vigorously for about 30 seconds, causingpersistent foam to appear over the liquid. The container was allowed tostand undisturbed for 5-6 hours to allow heavy particulates to settle.At the end of this time, a layer of foam had formed, while the liquiditself was divided into two strata: first, a narrow, very dark greenlayer resting on the bottom of the container below a second, muchlighter colored grayish-green and murky phase which represented perhaps85-90% of the total volume of liquid. The foam layer was carefullysiphoned off, using a capillary glass tube and vacuum suction. Thegrayish cloudy supernatant was then siphoned off, with care being takennot to disturb the dark bottom layer, which consisted mainly of settledcell bodies, and was transferred to a separate plastic container. Thegrayish cloudy supernatant was allowed to stand undisturbed for anadditional 16 hours. The liquid was initially almost colorless, lightgrey, but not transparent. After 16 hours settling time, a small amountof foam remained on top of the main body of liquid and a small amount ofgreen matter had settled on the bottom of the container. The liquid waslighter in color, but still not transparent. The foam on top of theliquid was siphoned off as before. The main body of liquid was thencarefully siphoned off, leaving behind the small amount of settled greenmaterial at the bottom of the container. The liquid which had thus beenisolated, contained the majority of the p-GlcNAc fibers and someimpurities.

[0280] To remove proteins and other unwanted matter liberated by thediatoms during the preceding steps in the procedure from thefiber-containing liquid, the suspension of fibers and cell remnants waswashed with sodium dodecyl sulfate (SDS). Specifically, the necessaryvolume of a 20% SDS solution was added to make the final concentrationof the liquid 0.5% SDS by volume. The container holding the liquid wassealed, secured in a horizontal position on a shaking machine, andagitated for 24 hours at about 100 shakes a minute. Soon after shakingbegan, large flocs of white p-GlcNAc fibers appeared in the suspension,and a considerable amount of foam accumulated in the head space of thecontainers. At the end of the SDS washing, the contents of thecontainers were transferred to a Buchner filtration equipment providedwith a Supor-800 polyether sulfone filter membrane, with 0.8 micron poresize (Gelman, Inc.). The liquid was filtered with suction, and thep-GlcNAc fibers in the liquid were collected on the filter membrane. Thep-GlcNAc fibers collected on the filter membrane were then washedfurther. First, the fibers were washed with hot (70° C.) distilled,deionized H₂O, using three times the volume of the original suspension.With a water jet using distilled, deionized H₂O, the white fiber clumpscollected on the filter membrane of the Buchner filter were transferredto a Waring blender, and the fiber clumps were disintegrated with about10 short mixing bursts. The suspension of disintegrated fibers wastransferred to a Buchner filter funnel equipped with a polyether sulfonefilter membrane as described above, and the liquid was removed undersuction. The collected fibers were washed with 1000 ml of hot. (70° C.)1N HCl solution, and subsequently further washed with 1000 ml hot (70°C.) distilled, deionized H₂O. Finally, the fibers were washed with 1000ml 95% ethanol at room temperature, and filtered to dryness. The fibermembrane and the filter membrane supporting the fiber membrane were thendried in a drying oven at 58° C. for 20 minutes. The membrane andmembrane support was then placed in a desiccator for 16 hours. Themembrane was then carefully detached from the filter membrane.

[0281] Second, p-GlcNAc was purified by using the acidtreatment/neutralization method described, above, in Section 5.3.2.Specifically, the p-GlcNAc was processed as described earlier in thisSection, until prior to the SDS wash step, at which point the solutionwas neutralized to a pH of approximately 7.0 by the addition of a 2.9MTris solution. The p-GlcNAc yield from this particular purificationprocedure was 20.20 milligrams per liter of diatom culture, although, onaverage, approximately 60 milligrams per liter diatom culture areobtained. SEM micrographs of membranes formed during the purificationprocedure are shown in FIGS. 8 and 9A-9E.

9. EXAMPLE: p-GlcNAc DEACETYLATION

[0282] A p-GlcNAc membrane was suspended in an aqueous 50% NaOHsolution. The suspension was heated at 80° C. for 2 hours. The resultingdeacetylated membrane was dried and studied by scanning electronmicroscopy, as shown in FIG. 11.

10. EXAMPLE: p-GlcNAc BIOCOMPATIBILITY

[0283] In this Example, it is demonstrated that the p-GlcNAc of theinvention exhibits no detectable biological reactivity, as assayed byelution tests, intramuscular implantation in rabbits, intracutaneousinjection in rabbits, and systemic injections in mice.

10.1. Materials and Methods 10.1.1. Elution Test

[0284] Conditions for the elution test conformed to the specificationsset forth in the U.S. Pharmacopeia XXII, 1990, pp. 1415-1497 and to U.S.Pharmacopeia XXII, Supplement 5, 1991, pp. 2702-2703.

[0285] Cell culture: Mouse fibroblast L929 cell line (American TypeCulture Collection Rockville, Md.; ATCC No. CCL1; NCTC clone 929) wasutilized. A 24 hour confluent monolayer of L929 cells was propagated incomplete Minimum Essential Medium (MEM).

[0286] p-GlcNAc: a solid membrane of p-GlcNAc which had been preparedaccording to the Mechanical Force method of purification described,above, in Section 5.3.1, was extracted in 20 ml serum-supplemented MEMas per U.S. Pharmacopeia XXII (1990) requirements.

[0287] Controls: Natural rubber was used as a positive control, andsilicone was used as a negative control. Controls were tested in thesame manner as the test article, p-GlcNAc.

[0288] Extracts: Extracts were prepared at 37° C., in a humidifiedatmosphere containing 5 carbon dioxide, for 24 hours. Extracts wereevaluated for a change in pH, and adjustments were made to bring the pHto within +/−0.2 pH units of the original medium. Adjustments were madewith HCl to lower the extract pH or with NaHCO₃ to raise the extract pH.Extracts were sterile filtered by passage through a 0.22 micron filter,prior to being applied to the cell monolayer.

[0289] Dosing: 3 mls of p-GlcNAc or control extracts were used toreplace the maintenance medium of cell cultures. All extracts weretested in duplicate.

[0290] Evaluation Criteria: Response of the cell monolayer was evaluatedeither visually or under a microscope. The biological reactivity, i.e.,cellular degeneration and/or malformation, was rated on a scale of 0 to4, as shown below. The test system is suitable if no signs of cellularreactivity (Grade 0) are noted for the negative control article, and thepositive control article shows a greater than mild reactivity (Grade 2).The test article (i.e., p-GlcNAc) meets the biocompatibility test ifnone of the cultures treated with the test article show a greater thanmild reactivity. Grade Reactivity Description of Reactivity Zone 0 NoneDiscrete intracytoplasmic granules; No cell lysis 1 Slightly Not morethan 20% of the cells are round, loosely attached, and withoutintra-cytoplasmic granules; occasional lysed cells are present 2 MildNot more than 50% of the cells are round and devoid of intracytoplasmicgranules; extensive cell lysis and empty areas between cells 3 ModerateNot more than 70% of the cell layers contain rounded cells and/or arelysed 4 Severe Nearly complete destruction of the cell layers

10.1.2. Intramuscular Implantations

[0291] Animals: Healthy, New Zealand White Rabbits, male and female,(Eastern Rabbit Breeding Laboratory, Taunton, Mass.) were used. Rabbitswere individually housed using suspended stainless steel cages. Uponreceipt, animals were placed in quarantine for 8 days, under the sameconditions, as for the actual test. Hardwood chips (Sani-chips™, J.P.Murphy Forest Products, Montvale, N.J.) were used as non-contact beddingunder cages. The animal facility was maintained at a temperature of68°+/−3° F., with a relative humidity at 30-70%, a minimum of 10-13complete air exchanges per hour, and a 12-hour light/dark cycle usingfull spectrum fluorescent lights. Animals were supplied with commercialfeed (Agway ProLab, Waverly, N.Y.) under controlled conditions andmunicipal tap water ad libitum. No known contaminants were present inthe feed, bedding, or water which would be expected to interfere withthe test results. Animals selected for the study were chosen from alarger pool of animals. Rabbits were weighted to nearest log andindividually identified by ear tattoo.

[0292] p-GlcNAc: The p-GlcNAc used was as described, above, in Section10.1.1.

[0293] Implantation Test: Two rabbits were used for each implantationtest. On the day of the test, the animal skin on both sides of thespinal column was clipped free of fur. Each animal was anesthetized toprevent muscular movement. Using sterile hypodermic needles and stylets,four strips of the test p-GlcNAc (1 mm×1 mm×10 mm) were implanted intothe paravertebral muscle on one side of the spine of each of two rabbits(2.5 to 5 cm from the midline, parallel to the spinal column, and about2.5 cm from each other). In a similar fashion, two strips of the USPnegative control plastic RS (1 mm×1 mm×10 mm) were implanted in theopposite muscle of each animal. Animals were maintained for a period of7 days. At the end of the observation period, the animals were weighedand euthanized by an injectable barbiturate, Euthanasia-5 (VeterinaryLaboratories, Inc., Lenexa, Kans.) Sufficient time was allowed to elapsefor the tissue to be cut without bleeding. The area of the tissuesurrounding the center portion of each implant strip was examinedmacroscopically using a magnifying lens. Hemorrhaging, necrosis,discolorations and infections were scored using the following scale:0=Normal, 1=Mild, 2=Moderate, and 3=Severe. Encapsulation, if present,was scored by first measuring the width of the capsule (i.e., thedistance from the periphery of the implant to the periphery of thecapsule) rounded to the nearest 0.1 mm. The encapsulation was scored asfollows: Capsule Width Score None 0 up to 0.5 mm 1 0.6-1.0 mm 2 1.1-2.0mm 3 Greater than 2.0 mm 4

[0294] The differences between the average scores for the p-GlcNAc andthe positive control article were calculated. The test is considerednegative if, in each rabbit, the difference between the average scoresfor each category of biological reaction for the p-GlcNAc and thepositive control plastic implant sites does not exceed 1.0; or, if thedifference between the mean scores for all categories of biologicalreaction for each p-GlcNAc article and the average score for allcategories for all the positive control plastic implant sites does notexceed 1.0, for not more than one of four p-GlcNAc strips.

10.1.3. Intracutaneous Injections

[0295] Animals: New Zealand white rabbits were used and maintained asdescribed, above, in Section 10.1.2.

[0296] p-GlcNAc: A solid membrane of p-GlcNAc which had been preparedaccording to the mechanical force method of purification described,above, in Section 5.3.1, was placed in an extraction flask, to which 20ml of the appropriate medium were added. Extractions were performed byheating to 70° C. for 24 hours. Following this procedure, extracts werecooled to room temperature. Each extraction bottle was shaken vigorouslyprior to administration.

[0297] Intracutaneous Test: On the day of the test, animals were clippedfree of fur on the dorsal side. A volume of 0.2 ml of each p-GlcNAcextract was injected intracutaneously at five sites on one side of eachof two rabbits. More than one p-GlcNAc extract was used per rabbit. Atfive sites on the other side of each rabbit, 0.2 ml of the correspondingcontrol was injected. Injection sites were observed for signs oferythema, edema, and necrosis at 24, 48, and 72 hours after injection.Observations were scored according to the Draize Scale for the ScoringSkin Reaction (USP Pharmacopeia XXII, 1990, 1497-1500; USP PharmacopeiaXXII, Supplement 5, 1991, 2703-2705) as shown in Table II, below: TABLEII Draize Scale for Scoring Skin Reactions Value Erythema and EscharFormation No erythema 0 Very slight erythema (barely perceptible) 1 Welldefined erythema 2 Moderate to severe erythema 3 Severe erythema (beetredness) to slight eschar 4 formation (injuries in depth) Total possibleerythema score = 4 Edema Formation No edema 0 Very slight edema (barelyperceptible) 1 Slight edema (edges are well defined by definite 2raising) Moderate edema (raised approximately 1 mm and 3 extendingbeyond area of exposure) Severe edema (raised more than 1 mm and 4extending beyond area of exposure) Total possible edema score = 4

[0298] All erythema and edema scores at 24, 48, and 72 hours weretotaled separately and divided by 12 (i.e., 2 animals×3 scoringperiods×2 scoring categories) to determine the overall mean score forthe p-GlcNAc versus the corresponding control. Animals were weighed atthe end of the observation period and euthanized by injection of abarbiturate, Euthanasia-5 (Veterinary Laboratories, Inc., Lenexa,Kans.). The results of the test are met if the difference between thep-GlcNAc and the control means reaction scores (erythema/edema) is 1.0or less).

10.1.4. Systemic Injections

[0299] Animals: Albino Swiss mice (Mus musculus), female, (Charles RiverBreeding Laboratories, Wilmington, Mass.) were used. Groups of 5 micewere housed in polypropylene cages fitted with stainless steel lids.Hardwood chips (Sani-chips™, J.P. Murphy Forest Products, Montvale,N.J.) were used as contact bedding in the cages. The animal facility wasmaintained as a limited access area. The animal rooms were kept at atemperature of 68+/−3° F., with a relative humidity of 30-70%, a minimumof 10-13 complete air exchanges per hour, and a 12 hour light/dark cycleusing full spectrum fluorescent lights. Mice were supplied withcommercial feed and municipal tap water ad libitum. There were no knowncontaminants present in the feed, bedding, or water which would beexpected to interfere with the test results. Animals selected for thestudy were chosen from a larger pool of animals. Mice were weighed tothe nearest 0.1 g and individually identified by ear punch.

[0300] p-GlcNAc: The samples used were as described, above, in Section10.1.1. Extracts were prepared according to the procedures described inSection 10.1.3, above.

[0301] Systemic Injection Test: Groups of 5 mice were injected witheither p-GlcNAc extract or a corresponding control article, in the sameamounts and by the same routes as set forth below: Test Article orControl Article Injection Extracts Dosing Route Dose/Kg Rate 0.9% SodiumIntravenous 50 ml 0.1 ml/sec Chloride Injection, USP (0.9% NaCl) 1 in 20Alcohol Intravenous 50 ml 0.1 ml/sec in 0.9% Sodium Chloride InjectionUSP (EtOH:NaCL) Polyethylene Intraperitoneal 10 g — Glycol 400 (PEG 400)Cottonseed Oil Intraperitoneal 50 ml — (CSO)

[0302] The animals were observed immediately after injection, at 24hours, 48 hours, and 72 hours after injection. Animals were weighed atthe end of the observation period and euthanized by exposure to carbondioxide gas. The requirements of the test are met if none of the animalstreated with the p-GlcNAc shows a significantly greater biologicalreactivity than the animals treated with the control article.

10.2. Results 10.2.1. Elution Test

[0303] The response of the cell monolayer to the p-GlcNAc test articlewas evaluated visually and under a microscope. No cytochemical stainswere used in the evaluation. No signs of cellular biological reactivity(Grade 0) were observed by 48 hours post-exposure to the negativecontrol article or to the p-GlcNAc. Severe reactivity (Grade 4) wasnoted for the positive control article, as shown below in Table III:TABLE III REACTIVITY GRADES Control Articles p-GlcNAc Negative PositiveTime A B A B A B  0 Hours 0 0 0 0 0 0 24 Hours 0 0 0 0 4 4 48 Hours 0 00 0 4 4

[0304] The p-GlcNAc of the invention, therefore, passes requirements ofthe elution test for biocompatability, and, thus, is non-cytotoxic.

10.2.2. Intramuscular Implantations

[0305] Both rabbits (A and B) tested increased in body weight andexhibited no signs of toxicity. See Table IV for data. In addition,there were no overt signs of toxicity noted in either animal.Macroscopic evaluation of the test and control article implant sitesshowed no inflammation, encapsulation, hemorrhage, necrosis, ordiscoloration. See Table IV for results. The test, therefore,demonstrates that the p-GlcNAc assayed exhibits no biologicalreactivities, in that, in each rabbit, the difference between theaverage scores for all of the categories of biological reaction for allof the p-GlcNAc implant sites and the average score for all categoriesfor all the control implant sites did not exceed 1.0. TABLE IVIMPLANTATION TEST (Macroscopic Observations) Test Article: p-GlcNAcAnimal Species: Rabbit TISSUE TEST CONTROL SITE: T1 T2 T3 T4 AVERAGE C1C2 AVERAGE Animal #: A Inflammation 0 0 0 0 0 0 0 0 Encapsulation 0 0 00 0 0 0 0 Hemorrhage 0 0 0 0 0 0 0 0 Necrosis 0 0 0 0 0 0 0 0Discoloration 0 0 0 0 0 0 0 0 Total 0 0 0 0 0 0 MEAN 0 0 0 0 0 0 SCORE:(total/5) AVERAGE 0 CONTROL VALUE: Animal #: B Inflammation 0 0 0 0 0 00 0 Encapsulation 0 0 0 0 0 0 0 0 Hemorrhage 0 0 0 0 0 0 0 0 Necrosis 00 0 0 0 0 0 0 Discoloration 0 0 0 0 0 0 0 0 Total 0 0 0 0 0 0 MEAN 0 0 00 0 0 SCORE: (total/5) AVERAGE 0 CONTROL VALUE:

10.2.3. Intracutaneous Test

[0306] All of the animals increased in weight. See Table V for data.There were no signs of erythema or edema observed at any of the p-GlcNAcor control article sites. Overt signs of toxicity were not observed inany animal. Because the difference between the p-GlcNAc and controlarticle mean reaction scores (erythema/edema) was less than 1.0, thep-GlcNAc meets the requirements of the intracutaneous test. See Table VIfor results. Therefore, as assayed by this test, the p-GlcNAcdemonstrates no biological reactivity. TABLE V Intracutaneous andImplant Tests Body Weights and Clinical Observations Test Article:p-GlcNAc Animal Species: Rabbit Body Weight (Kg) Weight Signs of GroupAnimal # Sex Day 0 Day 3 Change Toxicity* 0.9% 23113 Male 2.51 2.55 0.04None NaCl & CSO 0.9% 23114 Female 2.43 2.46 0.03 None NaCl & CSO EtOH:23115 Male 2.47 2.50 0.03 None NaCl & PEG 400 EtOH: 23116 Female 2.592.63 0.04 None NaCl & PEG 400 Day 0 Day 7 Implant A Male 2.74 2.80 0.06None B Female 2.66 2.74 0.08 None

[0307] TABLE VI INTRACUTANEOUS TEST DRAIZE SCORES Animal SITE NUMBERSSCORING (ER/ED) Averages ID # Vehicle T-1 C-1 T-2 C-2 T-3 C-3 T-4 C-4T-5 C-5 Time: T C NaCl Extract 23113 NaCl 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23114 NaCl 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 CSOExtract 23113 CSO 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 23114 CSO 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 NaCl/EtOH Extract 23115 NaCl 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 EtOH 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/072 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/023116 NaCl 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 EtOH0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 PEG Extract 23115 PEG 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23116 PEG 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

10.2.4. Systemic Test

[0308] All of the mice treated with the p-GlcNAc extract or the controlarticle increased in weight. See Table VII for data. In addition, therewere no overt signs of toxicity observed in any p-GlcNAc or controlanimal. See Table VI for results. It is concluded, therefore, that noneof the p-GlcNAc test animals showed a significantly greater biologicalreactivity than the animals treated with the control article. TABLE VIIANIMAL WEIGHTS AND CLINICAL OBSERVATIONS Body Weight (g) Dose Day DayWeight Signs of Group Sex (ml) Animal # 0 3 Change Toxicity* NaCl:Female 1.03 I. 20.6 22.8 2.2 None EtOH Female 1.06 II. 21.1 23.4 2.3None Test Female 1.02 III. 20.4 22.6 2.2 None 50 Female 1.11 IV. 22.224.5 2.3 None ml/kg Female 1.05 V. 21.0 23.2 2.2 None Mean 21.1 23.3 SD+/− 0.7 0.7 NaCl: Female 1.04 VI. 20.7 23.2 2.5 None EtOH Female 1.04VII. 20.8 23.5 2.7 None Control Female 1.02 VIII. 20.3 22.3 2.0 None 50Female 0.91 IX. 18.2 20.6 2.4 None ml/kg Female 0.94 X. 18.7 20.9 2.2None Mean 19.7 22.1 SD +/− 1.2 1.3 PEG Female 1.02 XI. 20.3 22.7 2.4None Test Female 0.96 XII. 19.2 21.4 2.2 None 10 Female 0.95 XIII. 18.921.6 2.7 None ml/kg Female 1.05 XIV. 20.9 22.7 1.8 None Female 0.94 XV.18.7 21.2 2.5 None Mean 19.6 21.9 SD +/− 1.0 0.7 PEG Female 1.01 XVI.20.1 22.3 2.2 None Control Female 0.99 XVII. 19.8 22.0 2.2 None 10Female 1.10 XVIII. 22.0 24.3 2.3 None g/kg Female 1.07 XIX 21.4 23.6 2.2None Female 1.03 XX. 20.6 22.4 1.8 None Mean 20.8 22.9 SD +/− 0.9 1.0

11. EXAMPLE: p-GlcNAc REFORMULATION

[0309] In the Working Example presented in this Section, a p-GlcNAcmembrane (16.2 mg) was dissolved in 1 ml of a dimethylacetamide solutioncontaining 5% LiCl. The p-GlcNAc-containing solution was placed in asyringe and extruded into 50 ml of pure water to precipitate a fiber.The resulting fiber was studied with scanning electron microscopy, asshown in FIG. 10.

12. EXAMPLE: CELL ATTACHMENT TO p-GlcNAc

[0310] In this working example, it is demonstrated that p-GlcNAcrepresents an efficient substrate for cell attachment and growth inculture.

12.1. Materials and Methods

[0311] Cells: The transformed mouse 3T3 fibroblast cell line was used,and was grown in DMEM supplemented with 10 fetal bovine serum (FBS).

[0312] p-GlcNAc membranes: p-GlcNAc was prepared according to themethods described, above, in Sections 5.3.1 and 8.

[0313] p-GlcNAc membranes were initially stuck to a #1 (18 mm) Corningcover glass using one drop of water, and were attached by autoclaving at121° C. for 30 minutes. Membranes prepared in this manner were thenplaced in culture wells of 6 well culture plates.

[0314] Cell counts: Cell numbers were determined in media by directcounting with a hemocytometer, and on matrix by first rinsing membraneswith fresh medium DMEM+10% FBS) followed by treatment with trypsin (10%,at 37° C. for 5 minutes) prior to counting.

[0315] SEM operating conditions: A Zeiss 962 instrument was utilizedwith an accelerating voltage of 10 kv, and a working distance of 15 mm.Polaroid type 55 p/n (u4) was utilized at various magnifications, asindicated. Sample coat:carbon coat (100{dot over (a)}) & 100{dot over(a)} aupd.

[0316] Specimen preparation: For primary fixation, the culture growthmedium was replaced with 2% glutaraldehyde in Eagle's DMEM withoutserum. Several changes were performed to ensure a complete transitionfrom growth media to fixative. Fixation proceeded for 0.5 hours at roomtemperature. Cover slips were transferred to fresh vials containing 2%Glutaraldehyde in 0.1M Na Cacodylate at pH 7.2 with 0.1M Sucrose andfixed for a further 1.5 hours at room temperature.

[0317] Dehydration, CPD, Mount and Sputter Coating:

[0318] Samples were rinsed in 0.1M Na Cacodylate pH 7.2, and cover slipswere transferred to a CPD holder. Dehydration was performed in ethanolseries (30%, 50%, 75%, 85%, 95% and 3×100%, 5 mins each), and sampleswere critical point dried. Cover slips were then mounted on Al stubs,carbon coated, using vacuum evaporator (@100{dot over (A)}) and sputtercoated with 100 {dot over (A)} AuPd.

12.2. Results

[0319] p-GlcNAc membranes were tested for an ability to form a substrateon which cells may be grown in culture. Mouse fibroblast cells weregrown in wells in the presence or absence of p-GlcNAc membranes and cellcounts were taken daily to assay the viability of cultures. The resultsof one such series of cell counts is shown in FIG. 14. As indicated, byday 5 after plating, only the wells containing p-GlcNAc membranes wereable to continue to sustain viable cells, demonstrating that p-GlcNAcmembranes are capable of acting as efficient substrates for thecontinued growth of cells in culture.

[0320] Further, the SEM micrographs depicted in FIG. 15 show healthycells strongly attached to p-GlcNAc membranes.

13. EXAMPLE: p-GlcNAc/COLLAGEN HYBRIDS

[0321] Presented in this Working Example is the formation andcharacterization of a p-GlcNAc/collagen hybrid material.

13.1. Materials and Methods

[0322] Materials: Bovine Type I collagen was used in preparation of thehybrids described in this study. p-GlcNAc was prepared according to themechanical force method described, above, in Section 5.3.2.

[0323] Hybrid preparation: Collagen (10 milligrams/ml) and p-GlcNAc(0.25 milligrams/ml) aqueous suspensions were mixed, in differentratios, frozen in liquid N₂ (−80° C.), held at −9° C. for 4 hours, andlyophilized. Material was dehydrothermally cross-linked under vacuum(approximately 0.030 Torr) at 60° C. for 3 days.

[0324] Cell Culture: Mouse 3T3 fibroblast cells were grown on thecollagen/p-GlcNAc hybrids produced. Standard culturing procedures werefollowed, and SEM micrographs were taken after 8 days in culture.

13.2. Results

[0325] Collagen and p-GlcNAc aqueous suspensions were mixed in differingratios (namely, 3:1, 1:1, 2:2, and 1:3 collagen:p-GlcNAc suspensionratios), frozen, lyophilized, and crosslinked. Such a procedure yieldedcollagen/p-GlcNAc slabs. SEM micrographs of the resulting materials areshown in FIGS. 16B-E. FIG. 16A represents a collagen-only controlmaterial. Note the porous structure of the hybrid material.

[0326] The collagen/p-GlcNAc hybrids of the invention provide anefficient three-dimensional structure for the attachment and growth ofcells, as shown in the SEM micrographs in FIGS. 17A-D.

14. EXAMPLE: NMR CHARACTERIZATION OF PURE PREPARATIONS OF p-GlcNAc

[0327] Presented in this Example is an NMR (nuclear magnetic resonance)analysis of a pure p-GlcNAc preparation.

14.1. Materials and Methods

[0328] p-GlcNAc preparations: The p-GlcNAc used in the NMR studiesdescribed here was prepared using the chemical purification methoddescribed, above, in Section 5.3.2, with hydrofluoric acid utilized asthe chemical reagent.

[0329] NMR techniques: Solid state NMR data was obtained using a Bruker50OMH NMR spectrometer. Computer image analysis was used to transformthe raw NMR spectrum data so as to eliminate background and to normalizebaselines. An example of such transformed data are shown in FIG. 18.Transformed NMR curves such as that in FIG. 18 were used to obtain areasfor every carbon atom type, and then to calculate the ratios ofCH3(area) to C-atom(area). Such values, obtained as described, areprovided in FIG. 20.

14.2. Results

[0330] Solid state NMR data was obtained by measuring the C¹³-NMRspectrum of a 500 mg sample of p-GlcNAc. A typical NMR spectrum is shownin FIG. 19. The individual peaks represent the contribution to thespectrum of each unique carbon atom in the molecule. The relativepercentage of each type of carbon atom in the molecule was determineddividing the area of the peak generated by that carbon species by thetotal sum of the areas under all of the NMR peaks obtained in thespectrum. Thus; it was possible to calculate the ratio of each of theatoms of the molecule measured by a reference atom. All p-GlcNAcmolecules consist of N-acetylated glucosamine residues having C1, C2,C3, C4, C5 and C6 atoms, by definition. The ratio, then, of the area ofthe N-acetyl CH3 carbon atom peak to the areas of any of the glucosamineresidue carbon atom peaks, above, should be 1.0 if all of theglucosamine residues in the polymer are N-acetylated. Data such as thosein FIG. 20 were used to obtain values for the CH3 (area) ratios.

[0331] The calculated ratios in FIG. 20 are in many cases equal to ornearly equal to 1.0, within experimental error, e.g. CH3/C2=1.097,CH3/C6=0.984, CH3/C5=1.007, CH3/C1=0.886. These results are consistentwith the conclusion that the p-GlcNAc material of the invention is freeof contaminants and is fully acetylated (i.e. that essentially 100% ofthe glucosamine residues are N-acetylated).

15. EXAMPLE: SYNTHESIS AND BIOLOGICAL CHARACTERIZATION OF CONTROLLEDPORE SIZE THREE-DIMENSIONAL p-GlcNAc MATRICES

[0332] Described below, are methods for the production ofthree-dimensional p-GlcNAc based porous matrices having controlledaverage pore sizes. Such matrices have a variety of importantapplications, particularly, for example, as means for the encapsulationof cells. Such cell encapsulation compositions are useful astransplantable cell-based therapeutics, and in other cell & tissueengineering applications such as in cartilage regeneration. Thecapability to manipulate the morphology and dimensionality of p-GlcNAcmaterials, as demonstrated here, provides a powerful tool in expandingthe potential applications of the p-GlcNAc material of the invention.

15.1. Materials and Methods

[0333] p-GlcNAc starting material: p-GlcNAc was prepared using thechemical purification method described, above, in Section 5.3.2, withhydrofluoric acid utilized as the chemical reagent.

[0334] Matrix formation: Suspensions (5 mls) containing 20 mg p-GlcNAcsamples were made in the solvents listed below in Section 15.2, prior tolyophilization. Samples were then poured into wells of tissue culturedishes and frozen at −20° C. The frozen samples were then lyophilized todryness, and the resulting three-dimensional, matrices were removed.

[0335] Scanning electron microscopy techniques: The procedures utilizedhere were performed as described, above, in Section 12.1. The imagesshown in FIGS. 21A-G. are 200× magnifications of the matrix material,and a scale marking of 200 microns is indicated on each of thesefigures.

15.2. Results

[0336] p-GlcNAc suspensions were obtained with each of the followingsolvents, as described, above, in Section 15.1:

[0337] A. Distilled water

[0338] B. 10% methanol in distilled water

[0339] C. 25% methanol in distilled water

[0340] D. Distilled water only

[0341] E. 10% ethanol in distilled water

[0342] F. 25% ethanol in distilled water

[0343] G. 40% ethanol in distilled water

[0344] Samples of matrix formed using each of the solvents weresubjected to scanning electron microscopic (SEM) analysis, as shown inFIGS. 21A-G. These figures reveal that the average matrix pore sizedecreases as the percentage of either methanol or ethanol increases ineach suspension.

[0345] Specifically, pore diameter in the two water suspensions (FIGS.21A and 21D) approach 200 microns on average. Pore size in the samplesdepicted in FIGS. 21C and 21F (25% methanol and ethanol, respectively)are between 30 and 50 microns on average.

[0346] The results shown here suggest that while both ethanol andmethanol may be successfully used to control p-GlcNAc pore size, ethanolmay be more efficient than methanol.

16. EXAMPLE: CELL GROWTH ON THREE-DIMENSIONAL POROUS p-GlcNAc MATRICES

[0347] Described in this Section are results demonstrating thesuccessful use of three-dimensional p-GlcNAc porous matrices assubstrates for the culturing of cells.

16.1. Materials and Methods

[0348] p-GlcNAc starting material: p-GlcNAc was prepared using thechemical purification method described, above, in Section 5.3.2, withhydrofluoric acid utilized as the chemical reagent.

[0349] Matrix formation: Three-dimensional p-GlcNAc matrices wereprepared by the lyophilization of suspensions of p-GlcNAc in water,water-ethanol, or water-methanol mixtures.

[0350] Suspensions (5 mls) containing 20 mgs p-GlcNAc were prepared inthe following solvents prior to lyophilization:

[0351] 1. Distilled water only

[0352] 2. 10% methanol in distilled water

[0353] 3. 25% methanol in distilled water

[0354] 4. Distilled water only

[0355] 5. 10% ethanol in distilled water

[0356] 6. 25% ethanol in distilled water

[0357] 7. 40% ethanol in distilled water

[0358] Samples were poured into circular wells of plastic tissue culturedishes and were frozen at −20° C. The frozen samples were thenlyophilized to dryness, and the resulting three-dimensional matriceswere removed. Samples of each matrix were subjected to scanning electronmicroscopic (SEM) analysis.

[0359] Cells: Mouse embryo BALBC/3T3 fibroblast cell line (clone A31),obtained from the ATCC, were used for culturing on the three-dimensionalporous p-GlcNAc matrices.

[0360] Culturing conditions: One cm² samples of porous matrices wereplaced in tissue culture wells and were covered with a standardtissue-culture growth medium. Each well was seeded and cells werecultured for 6 days at 37° C. in a CO₂ incubator (5% CO₂)

[0361] SEM procedures: Matrix samples were fixed and subjected to SEManalysis as described, above, in Section 12.1. The matrices wereprepared by lyophilizing p-GlcNAc in distilled water. Images vary inmagnification from 100× to 5000×, as indicated in figure legends (FIGS.22A-G).

16.2. Results

[0362] SEM photographs of p-GlcNAc matrices containing attached mousefibroblast cells attached are shown in FIGS. 22A-G. These photographsshow that the three-dimensional p-GlcNAc matrices contain attached mousefibroblast cells. Further, the photographs reveal that there is a closeinteraction and connection between the cells and the p-GlcNAc matrixmaterial. It is also notable that the cells have a roundedthree-dimensional morphology which is different from the flat, spreadshape of the cells when cultured directly onto plastic culture dishes.Cell viabilities were determined to be greater than 95%.

17. EXAMPLE: p-GlcNAc SUCCESSFULLY REDUCES AND PREVENTS POST-SURGICALADHESIONS

[0363] The Example presented herein demonstrates the successful use ofp-GlcNAc materials, specifically a p-GlcNAc membrane and gelformulation, to reduce or prevent the formation of post-surgicaladhesions in a series of animal models for such adhesions.

17.1. Materials and Methods

[0364] Synthesis p-GlcNAc-lactate: p-GlcNAc membrane starting materialwas produced by the chemical method, as described, above, in Section5.3.2, with hydrofluoric acid utilized as the chemical reagent.

[0365] The p-GlcNAc was converted to deacetylated p-GlcNAc by thefollowing method. (It should be noted that approximately 1.4 g ofp-GlcNAc are needed to produce 1 g of p-GlcNAc lactate, given theexpected loss in mass of approximately 15% which occurs upondeacetylation). In a stoppered flask approximately 200 mg of p-GlcNAcmembrane material were mixed vigorously with approximately 200 ml 60%NaOH. The vigorous shaking served to separate the p-GlcNAc membranematerial to the greatest extent possible. The NaOH solution used wasmade at least 12 hours before use. The reaction flasks were placed in an80° C. water bath for 6 hrs, with periodic shaking to separate and wetthe p-GlcNAc material. After 6 hrs, the samples were taken from thewater bath and the NaOH solution was immediately decanted. The membranematerials were washed with ddH₂O, at room temperature, until a pH of 7was reached The membranes were removed from the water and dried on aTeflon sheet.

[0366] At this point a 2 mg sample was collected for C, H, N analysis inorder to determine the extent of deacetylation. Further, solubility ofthe deacetylated material in it acetic acid was checked, with asolubility of 1 mg/ml indicating that the p-GlcNAc material wasappropriately deacetylated.

[0367] The partially deacetylated pGlcNAc was then converted topGlcNAc-lactate using the following method: Sufficient 2-propanol(containing 10% water) to wet all of the partially deacetylated pGlcNAcmaterial and to allow for stirring was added to 1 g of the partiallydeacetylated p-GlcNAc in a 250 ml Erlenmeyer flask. (Approximately 30mls 2-propanol are required.) 2-propanol must be reagent grade, andfresh prior to each synthesis. With stirring, 1.1 mL of a 50% aqueouslactic acid solution is added. Lactic acid should be reagent grade, andmust be analyzed to determine the exact concentration of available(i.e., non-esterified) lactic acid present. This was generallyaccomplished by titration with 0.1N NaOH to the phenolphthaleinend-point (pH 7.0). The concentration of lactic acid used must beconstant, i.e., must be +/−1 percent, for each p-GlcNAc synthesis. Themixture was allowed to stir for at least two hours at room temperature.It is possible to add low heat in order to increase the reaction rate.Reaction time may be extended, or the amount of 50% aqueous lactic acidmay be increased to ensure that the reaction goes to completion. Thecontents of the flasks were finely filtered through a Buchner funnelusing quantitative ashless filter paper. The material was washed with 15ml of anhydrous 2-propanol. The material was allowed to air dry in afume hood for 2 hours and then placed in an oven at 40° C. overnight.For every gram of partially deacetylated p-GlcNAc starting material, afinal p-GlcNAc-lactate weight of approximately 1.4 g, (i.e., an increaseof 40% in mass) should be obtained.

[0368] Formulation of p-GlcNAc-lactate as a gel: The p-GlcNAc-lactatematerial was formulated into a gel as follows: p-GlcNAc-lactate materialwas dissolved in distilled and deionized water to a concentration ofbetween 0.1-4.0% p-GlcNAc-lactate, by weight. Reagent grade propyleneglycol (2-propanediol) was then added to a final propylene glycolconcentration of between 1-10%. In some cases, a preservative was addedto prevent bacterial and/or fungal contamination of the product.Typically, concentrations of p-GlcNAc-lactate of between 0.1-4.0% wereprepared. The viscosity of these preparations increases dramatically asthe p-GlcNAc-lactate, percentage increases, such that formulationshaving 0.5% or more of the p-GlcNAc-lactate behave as gels.

[0369] Animal Models:

[0370] Sprague-Dawley rats: Adhesions are produced in this model byabrading or irritating the serosal surface of the cecum and apposing itto an area of parietal peritoneum. The success rate for inducingadhesions in control animals with this method is reported to be at anaverage 80%.

[0371] Specifically, the surgical procedure for inducing post-surgicaladhesions in these rats involved the following. Animals were placed indorsal recumbency and prepared and draped accordingly for asepticsurgery. Abdominal cavities were exposed through a midline incision. Anarea, approximately 0.5 cm×1.0 cm, of parietal peritoneum on the leftabdominal wall was carefully excised, removing a thin layer of muscle,along with the peritoneum.

[0372] The cecum was then elevated and isolated. An area, approximately0.5 cm×1.0 cm, on the lateral surface of the proximal end of the cecumwas abraded by rubbing ten times with a dry gauze. The cecum was thenscraped with a scalpel blade to cause minute petechial hemorrhages. Thececal abrasion and the peritoneal incision were left exposed for 15minutes.

[0373] After 15 minutes, the test article (i.e., the p-GlcNAc material)or control article was applied to the cecal wound. The cecal abrasionand the peritoneal wound were then apposed and held in contact withAllis tissue forceps for an additional 15 minutes.

[0374] The cecum was then replaced into the abdomen such that theabraded area of the cecum was adjacent to the peritoneal site. Theabdominal incision was closed and the animal was allowed to recover fromthe anesthesia.

[0375] Fourteen days after surgery, animals were euthanized and theabraded area was examined for the formation of post-surgical adhesions.If adhesions were present, the entire area involved in the adhesion(i.e., body wall, test or control article, and internal organs) weredissected free of the animal.

[0376] The extent of involvement and tenacity of adhesions wereevaluated according to the following scales:

[0377] Extent of Involvement Scores:

[0378] 0 no adhesion

[0379] 1 adhesion <=25% of the area

[0380] 2 adhesion <=50% of the area

[0381] 3 adhesion <=75% of the area

[0382] 4 adhesion >75% of the area

[0383] Tenacity Scores:

[0384] 0 no adhesion

[0385] 1 adhesion freed with blunt dissection

[0386] 2 adhesion freed with aggressive dissection

[0387] 3 adhesion requiring sharp dissection

[0388] Additional animal models: Pig and horse large animal bowel modelswere used to assess the prevention of peritoneal adhesions.

[0389] Surgical procedure: The animals were placed in dorsal recumbencyand prepared and draped accordingly for aseptic surgery. The abdominalcavity was exposed through a midline incision. The small intestine waselevated and a 2 cm×2 cm section was identified, extensively abraded(approximately 200 strokes using a scalpel), and allowed to dry for 10minutes. The test article (i.e., p-GlcNAc material) or control articlewas then applied to the abraded wound, and the wounded section of thesmall intestine was replaced into the abdomen. In such a large boweltype of animal model, six wounds, each separated by 4 inches of bowelfrom the adjacent wound provides an environment highly prone to formadhesions. Following induction of the last of the wounds, the abdominalincision is closed and the animal is allowed to recover from theanesthesia.

[0390] Analysis of peritoneal adhesions: Twenty one days after surgery,animals were euthanized and the abraded area was examined, with adhesionformation being evaluated following a procedure similar to that of theSprague-Dawley rat cecum model.

17.2. Results

[0391] When injury occurs, the body sets in motion a complex set ofresponses designed to restore the injured area. In the final stages ofhealing, connective tissue forms at the wound site to regenerate thebody structure and protect the affected area from further damage. Insome instances this cascade of events does not work properly and canlead to life threatening conditions.

[0392] For example, as a visceral organ heals following surgery, afibrin clot generated during the surgical procedure may invade thesurface of adjoining organs forming a link which allows for fibroblastmigration. This migration leads to collagen deposition and tissuegrowth, which in turn causes the organs involved to adhere to oneanother.

[0393] Such adhesions, referred to as post-surgical adhesions, mayproduce pain, obstruction and malfunction by distorting the organ ororgans involved. Immobilized joints, intestinal obstruction andinfertility are often linked to the formation of post-surgicaladhesions. Furthermore, post-surgical adhesion will complicate andextend the length of future surgical procedures in the surroundingregion. This last issue is of particular relevance to open heartsurgeries and cesarean section obstetrical procedures where additionalsurgeries may be required. The formation of adhesions is very commonfollowing abdominal, cardiovascular and orthopedic surgical procedures.

[0394] When adhesions become pathological and seriously interfere withorgan function, surgical adhesiolysis (sharp or blunt dissection of theadhesion in conjunction with meticulous surgical techniques) is thetreatment that is currently used to eliminate adhesions. In 1991,approximately 500,000 adhesiolysis procedures were performed in the U.S.This procedure is, however, notoriously ineffective, with the frequencyof recurrence of adhesion formation reported to be as high as 90%.Further, no other technique or composition has proven effective in theprevention of such post-surgical adhesions.

[0395] The results presented herein, therefore, are significant in thatthey demonstrate the effectiveness of the p-GlcNAc materials of theinvention for the prevention of post-surgical adhesions. Specifically,the results presented here demonstrate the efficacy of p-GlcNAc basedsolid and liquid, formulations as barriers to the formation of abdominalpost-surgical adhesions in accepted rat and pig animal model systems.

[0396] One of the accepted animal models used to study adhesionformation employs visceral-parietal peritoneal adhesions inSprague-Dawley rats. Both partially deacetylated p-GlcNAc membranes andp-GlcNAc-lactate gel formulations prevented and/or considerably reducedthe incidence of adhesion formation as compared with either non-treatedcontrols or treated with InterCEED™ (Johnson & Johnson), the onlycommercially available product for this indication.

[0397] Specifically, a total of 18 rats were used to testp-GlcNAc-lactate gel formulations. 12 animals were used as controls,with 6 receiving no treatment and 6 receiving InterCeed™. 6 animalsreceived 0.25% p-GlcNAc-lactate gel, 10% propylene glycol, water.Animals receiving the p-GlcNAc-lactate gel treatment showed asignificantly reduced incidence of postoperative adhesion formation,compared to either of the controls, as shown, below, in Table VIII.TABLE VIII Extent of Involvement Tenacity Control (No treatment) 1 +/−2.1 1 +/− 1.5 InterCEED ™ 1 +/− 1.8 1 +/− 1.5 p-GlcNAc-lactate gel 0 +/−0.8 1 +/− 1.2

[0398] Partially deacetylated p-GlcNAc membranes were also tested fortheir ability to reduce or prevent the occurrence of post-surgicaladhesions in the rat animal model. A total of 22 rats were used in thestudy. 12 animals were used as controls, with 6 receiving no treatmentand 6 receiving InterCEED™. Ten animals each received a 1 cm×1 cmmembrane of an approximately 60% deacetylated p-glcNAc formulation. Theanimals which received the partially deacetylated p-GlcNAc membraneshowed a significant reduction in the incidence of formation ofpostoperative adhesions, as compared with the non-treated controls andInterCEED™, as shown, below, in Table IX. TABLE IX Extent of InvolvementTenacity Control (No treatment) 3 +/− 1.8 1 +/− 0.6 InterCEED ™ 3 +/−1.6 1 +/− 0.4 p-GlcNAc-membrane 1 +/− 0.8 1 +/− 0.3

[0399] Large animal bowel models for the prevention of peritonealadhesions were also used to test p-GlcNAc compositions. Specifically,six pigs and one horse were used to study both the partiallydeacetylated p-GlcNAc membrane and the p-GlcNAc-lactate gel. Thepartially deacetylated p-GlcNAc membrane consisted of a 2 cm×2 cm pieceof 60% deacetylated p-GlcNAc membrane, while the p-GlcNAc-lactate gelconsisted of 0.25% p-GlcNAc lactate formulated with 10% propylene glycoland water. Control animals received no treatment to the wounded site.

[0400] The results of these large animal studies revealed that, whilethe control sites formed multiple adhesions and scar tissue in thesurrounding site, both the p-GlcNAc membrane and gel formulationseffectively reduced the formation of adhesions.

[0401] Samples from control and treated sites were additionally examinedusing SEM, which showed an increased amount of fibrosis in the controlsites as compared to the treated tissues.

18. EXAMPLE: BIODEGRADABILITY OF p-GlcNAc MATERIALS

[0402] The Example presented in this Section demonstrates that p-GlcNAcmaterials of the invention may be prepared which exhibit controllable invitro and in vivo biodegradability and rates of resorption.

18.1. Materials and Methods

[0403] p-GlcNAc materials: Prototype I was made by the method described,above, in Section 5.3.2, via the chemical method, with hydrofluoric acidbeing utilized as the chemical reagent. Prototype I represented 100%acetylated p-GlcNAc.

[0404] The p-GlcNAc starting material of prototype 3A was made by themethod described, above, in Section 5.3.2, via the chemical method, withhydrofluoric acid being utilized as the chemical reagent. The p-GlcNAcmaterial was then deacetylated by the method described, above, inSection 5.4. Specifically, the p-GlcNAc material was treated with a 40%NaOH solution at 60° C. for 30 minutes. The resulting prototype 3A wasdetermined to be 30% deacetylated.

[0405] The p-GlcNAc starting material of prototype 4 was made by themethod described, above, in Section 5.3.2, via the chemical method, withhydrofluoric acid being utilized as the chemical reagent. The p-GlcNAcmaterial was then deacetylated by treatment with a 40% NaOH solution at60° C. for 30 minutes. Next, the fibers were suspended in distilledwater, frozen at −20° C., and lyophilized to dryness. Prototype 4 wasalso determined to be 30% deacetylated.

[0406] Abdominal implantation model: Sprague Dawley albino rats wereutilized for the abdominal implantation model studies. Animals wereanesthetized and prepared for surgery, and an incision was made in theskin and abdominal muscles. The cecum was located and lifted out. A 1cm×1 cm membrane of p-GlcNAc material was placed onto the cecum, and theincision was closed with nylon. Control animals were those in which nomaterial was placed onto the cecum.

[0407] Animals were opened at 14 and 21 days post implantation.Photographs Were taken during the implant and explant procedures (FIGS.23A-E). Samples of cecum were prepared for histopathology after theexplant procedure.

[0408] p-GlcNAc in vitro degradation lysozyme-chitinase assay: The assayis a calorimetric assay for N-acetyl glucosamine, and was performed asfollows: 150 μl of a reaction sample was pipetted into 13×100 mm glassdisposable test tubes, in duplicate. 25 μl of 0.25M potassium phosphatebuffer (pH 7.1) was added to each test tube, followed by the addition of35 μl of 0.8M potassium borate solution (pH 9.8). Tubes were immediatelyimmersed into an ice-bath for a minimum of 2 minutes. Samples were thenremoved from the ice-bath, 1 ml of freshly prepared DMAB reagent wasadded, and the samples were vortexed. DMAB (Dimethyl aminobenzaldehyde)reagent was made by adding 70 mls of glacial acetic acid and 10 mls of11.6N (concentrated) HCl to 8 grams of p-dimethyl aminobenzaldehyde.Samples were then incubated at 37° C. for 20 minutes.

[0409] To prepare a standard curve, the following procedure wasutilized. A GlcNAc stock solution was diluted to 0.1 mg/ml with 0.10Msodium acetate buffer (pH 4.5) and 0 μl, 20 μl, 30 μl, 90 μl or 120 μlof the diluted GlcNAc solution was added to a set of test tubes. Thiswas followed by the addition of 150 μl, 130 μl, 60 μl or 30 μl,respectively, of 0.010M sodium acetate buffer (pH 4.5) to the testtubes. Next, 25 μl of 0.25M potassium phosphate buffer (pH 7.1) and 35μl of 0.8M potassium borate buffer (pH 9.8) were added to each testtube. A duplicate set of test tubes is prepared by the same procedure.

[0410] The test tubes are capped and boiled at 100° C. for exactly 3minutes. The tubes are then immersed in an ice bath. The tubes areremoved from the ice bath and 1 ml of DMAB reagent, freshly preparedaccording to the method described above in the Section, is added to eachtube. The tubes are incubated at 37° C. for 20 minutes. The absorbanceof the contents of each tube is read at 585 nM. Absorbance should beread as quickly as possible. The standard curve is plotted on graphpaper and used to determine the concentration of N-acetyl glucosamine inthe reaction samples. A typical standard curve is shown in FIG. 23.

18.2. Results

[0411] The in-vitro biodegradability of p-GlcNAc materials was studiedin experiments which assayed the relative susceptibility of p-GlcNAcmembrane materials to degradation by lysozyme. p-GlcNAc membranes wereexposed to an excess of lysozyme in a 10 mM acetate buffer, and thesubsequent release of N-acetyl glucosamine was determined using theassay described, above, in Section 18.1.

[0412] The results of these experiments indicated that partiallydeacetylated membranes are more susceptible to digestion by lysozyme(see FIG. 24) and, further, that the rate of lysozyme degradation isdirectly related to the extent of deacetylation (see FIG. 25, whichcompares the degradation rates of a 50% to a 25% deacetylated p-GlcNAcmembrane).

[0413] p-GlcNAc In Vivo Degradation

[0414] Additionally, experiments were performed which addressed thein-vivo biodegradability of p-GlcNAc materials. Such experimentsutilized an abdominal implantation model. Three p-GlcNAc materials, aslisted below, were tested.

[0415] p-GlcNAc Materials Tested:

[0416] 1) p-GlcNAc, fully acetylated (designated prototype 1);

[0417] 2) partially deacetylated p-GlcNAc membrane (designated prototype3A); and

[0418] 3) lyophilized and partially deacetylated p-GlcNAc membrane(designated prototype 4).

Results

[0419] The fully acetylated p-GlcNAc (prototype 1) was resorbed within21 days, as shown in FIGS. 26A-26C. The partially deacetylated p-GlcNAcmembrane (prototype 3A) was completely resorbed within 14 days, as shownin FIGS. 26D-26E. The lyophilized and partially deacetylated p-GlcNAcmembrane (prototype 4) had not yet been completely resorbed after 21days post-implantation.

[0420] Histopathology analyses showed that once the p-GlcNAc materialhas been resorbed there were no histological differences detectablebetween tissue samples obtained from the treated and from the controlanimals.

19. EXAMPLE: p-GlcNAc HEMOSTASIS STUDIES

[0421] The experiments described herein study the efficacy of thep-GlcNAc materials of the invention for the control of controllingbleeding is, further, compared against commercially available hemostaticproducts.

19.1. Materials and Methods

[0422] p-GlcNAc and control materials: partially deacetylated(approximately 70%) p-GlcNAc membranes were made using the chemicalseparation technique described, above, in Section 5.3.2, withhydrofluoric acid being utilized as the chemical reagent, and thetechniques described, above, in Section 5.4. 2 cm×1 cm pieces were used.p-GlcNAc-lactate gel (4% p-GlcNAc-lactate, formulated in propyleneglycol and water) was produced following the methods described, above,in Section 17.1. The control material utilized for the study of bleedingin the spleen and liver was Gelfoam™ (Upjohn Company). Gelfoam™ andAvitene™ (Medchem Products, Inc.) were the control materials used in thestudy of small blood vessel bleeding.

[0423] Test animals: New Zealand White rabbits were used. 3 animalsreceived two wounds in the spleen and one wound in the liver. 4 animalsreceived five surgical wounds to blood vessels of similar size in thecaudal mesenteric artery system.

[0424] Surgical preparation: The animals were anesthetized with ketamineHCl and Xylazine. The animals were placed in dorsal recumbency, and allthe hair from the abdomen was removed. The abdomen was then scrubbedwith povidone-iodine and 70% isopropyl alcohol and draped for asepticsurgery.

[0425] Liver/spleen surgical procedures: A midline incision was made andeither the spleen or liver was exteriorized and packed with moistsponges. A 3-4 mm diameter cork borer was used to make a circular woundof about 2 mm depth at one end of the organ. Once the splenic tissue wasremoved, a pre-weighed 4″×4″ gauze sponge was used to absorb all theblood lost from the splenic wound for a period of one minute. The spongewas re-weighed to quantify the amount of blood lost from that particularwound. The test animal was then treated by application to the wound ofone of the treated materials. The time until hemostasis and the amountof blood lost prior to hemostasis was recorded.

[0426] After hemostasis in the first wound was achieved, a second woundin the spleen and one wound in the liver were made following the sameprocedure.

[0427] Small blood vessel surgical procedure: A midline incision wasmade and the small bowel was exteriorized exposing the caudal mesentericartery system. The bowel was packed with moist sponges and five bloodvessels of about the same size were identified. A scalpel was used tomake a wound of about 1 mm depth at one of the vessels. A pre-weighed4″×4″ gauze sponge was used to absorb all the blood lost from the vesselwound for a period of one minute. The sponge was re-weighed to quantifythe amount of blood lost from that particular wound. The animal was thentreated by application to the wound of one of the treatment materials.The time until hemostasis occurred and the amount of blood lost prior tohemostasis were recorded.

[0428] After hemostasis in the first wound was achieved, four morewounds were made following the same procedure.

19.2. Results

[0429] p-GlcNAc materials were tested for their ability to controlbleeding in the spleen and liver of rat animal models. The p-GlcNAcmaterials tested were: 1) partially deacetylated (approximately 70%)p-GlcNAc; and 2) p-GlcNAc-lactate gel (4% p-GlcNAc-lactate, formulatedin propylene glycol and water). The effectiveness of these p-GlcNAcmaterials was compared to Gelfoam™ (Upjohn Company).

[0430] Each material was tested three times (twice in the spleen andonce in the liver). Both of the p-GlcNAc based materials exhibited aneffectiveness in controlling bleeding within the first minute afterapplication which was comparable to that of Gelfoam™. The p-GlcNAc basedmaterials have additional advantages. Specifically, the p-GlcNAcmaterials do not need to be held in place during the procedure, may beleft in the body, where they will be resorbed within two to three weeks(Gelfoam™ is not indicated for this purpose), are compatible with bothgeneral and minimally invasive surgical procedures.

[0431] Next, the efficacy of p-GlcNAc based materials in the control ofbleeding in small blood vessels was studied, and compared againstcommercially available hemostatic products.

[0432] Each material was tested five times (twice in one of the animalsand once in the other three animals). The p-GlcNAc membrane and gelformulations were easily applied to the site and controlled the bleedingwithin 2 minutes. Gelfoam™, which had to be held in place in order toperform its function achieved hemostasis within the same 2 minute rangeas the p-GlcNAc materials. Avitene™, a fibrous material made ofcollagen, was difficult to handle and required more than five minutes tocontrol the bleeding.

[0433] Thus, the results described herein demonstrate that the p-GlcNAcmaterials tested here represent effective, convenient hemostatic agents.

20. EXAMPLE: p-GlcNAc DRUG DELIVERY SYSTEMS

[0434] Described herein are studies demonstrating the successful use ofp-GlcNAc materials to deliver anti-tumor drugs to the site of malignantskin cancer and colon cancer tumors such that the delivered anti-tumordrugs exhibit a therapeutic impact upon the tumors.

20.1. Materials and Methods

[0435] p-GlcNAc-lactate drug delivery compositions: Mixtures of5′-fluorouracil (5′-FU) and p-GlcNAc-lactate were formulated as follows;0.5 mL of 5′-FU (50 mg/mL) was mixed with 0.5 mL of propylene glycol,and 2.0 mL of 4% p-GlcNAc-lactate was added and mixed. Thep-GlcNAc-lactate was produced using the techniques described, above, inSection 5.4. Even after extensive mixing, the 5′FU did not completelydissolve into the p-GlcNAc-lactate gel. Assuming complete mixing, thefinal concentration of 5′-FU would be 6.25 mg/mL.

[0436] Mixtures of mitomycin (Mito) and p-GlcNAc-lactate were formulatedas follows; 0.5 mg of Mito (lyophilized powder) were dissolved in 5 mlof propylene glycol, and 0.5 ml of the Mito solution was mixed with 0.5mL of MPT's 4% p-GlcN-lactate preparation to give a final Mitoconcentration of 0.2 mg/ml and a final p-GlcNAc-lactate concentration of2%. The materials were compatible, with the Mito dissolving easily intothe p-GlcNAc-lactate gel.

[0437] p-GlcNAc membrane 5′FU delivery compositions: Samples of5′-fluorouracil (5′-FU) were immobilized into disks of pure p-GlcNAcmembrane material produced using the chemical separation methoddescribed, above, in Section 5.3.2, with hydrofluoric acid beingutilized as the chemical reagent. Each disk described here had adiameter of 1.5 cm, as described here.

[0438] For the preparation of high dose (HD) disks, 0.64 mL of a 50mg/mL solution of 5′-FU was mixed with suspensions containingapproximately 8 mg of pure p-GlcNAc. The mixtures were allowed to standfor several hours to promote the absorption of 5′-FU into the p-GlcNAc,and were then dried at 55° C. for 3.5 hours. The resulting HD diskscontained a total of 32 mg 5′-FU, which is equivalent to approximatelytwice the normal total 14 day dose of 5′-FU typically given to a cancerpatient, normalized to the weight of the experimental mice based on thetypical dose of 5′-FU per Kg body weight for humans. It should be notedhere, and in the low dose concentrations described below, that theamount of 5′-FU contained in the disks was approximated and was basedonly on the amount of 5′-FU put into the suspensions.

[0439] Low dose (LD) 5′-FU containing p-GlcNAc disks were prepared inthe same manner, except that the LD disks contained 17 mg of 5′-FU, anamount equivalent to equal the normal total human dose for a 14 dayperiod, normalized to of 5′-FU per Kg body weight for humans.

[0440] Test Animals: For the 5′FU study, SCID (severe combinedimmunodeficiency) mice were inoculated with subcutaneous flankinjections of HT-29 colon cancer cells (ATCC; 1×10⁵ cells per inoculum)obtained by standard tissue culture methods, in order to produce HT-29colon cancer tumors. These injections led to palpable tumors which wereharvested in 14-21 days. Tumors were dissected and necrotic tissue wascut away. The HT-29 colon cancer tumors were sliced into 3×3×3 mmpieces.

[0441] The experimental SCID mice were anesthetized via intra-peritonealinjections with a standard dose of Avetin, and a slice of HT-29 coloncancer tumor was implanted onto the cecum of each mouse. Specifically,each mouse was surgically opened to expose its abdomen and the cecum waslocated, which was nicked with a scalpel to make a small incision. A3×3×3 mm tumor slice was sutured over the incision onto the cecum using5.0 silk sutures. The abdomen was then closed using Clay Adams staples.

[0442] All mice were caged singly and fed for two weeks. All mice werehealthy and none had obstructed colons at the end of the two weekperiod.

[0443] On day 14, each mouse was anesthetized, and its abdomen wasreopened. The growing tumors were measured (length and horizontaldimensions). Tumors were then treated with the p-GlcNAc/anti-tumor drugor were used as controls.

[0444] Six mice were used for the p-GlcNAc-lactate 5′FU study, and 15mice were used for the p-GlcNAc membrane 5′FU study.

[0445] For the mitomycin study, nine SCID mice were inoculated withsub-cutaneous injections of A431 squamous cell skin cancer cells (ATCC;1×10⁵ cells per inoculum). Tumors resulted in all mice within 14 days.

[0446] Treatments: For the p-GlcNAc-lactate 5′FU study, animals weretreated once daily by “painting” the 5′-fluorouracil (5′-FU) containingp-GlcNAc gel mixture onto the skin area over the tumor mass.Measurements of the tumor size were obtained daily. Control animalsincluded animals treated with p-GlcNAc alone, without 5′-FU, and animalswhich received no treatment.

[0447] For the p-GlcNAc membrane 5′FU study, the HT29 colon tumors inthe SCID mice were treated by surgically implanting disks of thedrug-containing p-GlcNAc membrane material directly onto their surface,after having allowed the tumor to grow on the colon for 14 days. Micewere sacrificed 14 days following the implant procedure. Measurements oftumor volumes were made immediately prior to implanting thedrug-containing p-GlcNAc membranes on day 0 and at the termination ofthe experiment on day 14. Control animals included ones treated with thep-GlcNAc membrane without 5′-FU, and controls which received notreatment. Additionally, two animals received daily systemic injectionsof 5′-FU in doses equivalent to the HD and LD regimen.

[0448] For the p-GlcNAc-lactate Mito study, animals were treated dailyas in the p-GlcNAc-lactate 5′-FU study, with 3 animals being treatedwith the Mito containing mixture. In addition, 3 animals were treatedwith p-GlcNAc minus Mito, 2 animals received no treatment, and 1 animalreceived propylene glycol.

20.2. Results 20.2.1. p-GlcNAc-LACTATE 5′FU

[0449] Experiments designed to study the effect of p-GlcNAc-lactate Mitodrug delivery systems on tumor size were conducted, as described, above,in Section 20.1.

[0450] The largest length and width dimension were measured for eachtumor and the cross-sectional area using these dimensions wascalculated. The cross-sectional area values are shown in Table X, below.TABLE X Animal # Treatment Day 0 Day 4 Day 11 Day 15 Tumor Size (cm²) 1CL + 5FU 63 90 168 156 2 CL + 5FU 48 56 70 88 3 CL Control 21 36 88 1084 CL Control 58 110 150 195, 30 5 Nothing 40 64 132 234 6 Nothing 28 42100 132 % Increase in Size 1 CL + 5FU 0 43 167 147 2 CL + 5FU 0 17 47 843 CL Control 0 71 319 414 4 CL Control 0 90 160 289 5 Nothing 0 61 232488 6 Nothing 0 48 253 366

[0451] The data comparing p-GlcNAc-lactate 5′FU treated animals withcontrols are shown in FIGS. 27-28. The data summarized in Table X andFIGS. 27-28 clearly suggest that the HT-29 subcutaneous tumors in therats treated with the 5′-FU containing p-GlcNAc-lactate gels have asignificantly retarded rate of growth compared to controls. Their growthhas been slowed 2.5-fold in comparison to the p-GlcNAc-lactate gelcontrols and 4-fold compared to the no treatment controls.

20.2.2. p-GlcNAc-Lactate Mito

[0452] Experiments designed to study the effect of p-GlcNAc-lactate 5′FUdrug delivery systems on tumor size were also conducted, as described,above, in Section 20.1.

[0453] The largest length and width dimensions were measured for eachtumor and the cross sectional area using these dimensions wascalculated. The cross-sectional area values were as shown in Table XI,below. TABLE XI Tumor Size (cm²) Animal # Treatment Day 0 Day 3 Day 5Day 8 1 pGlcNAc- 23 23 42 49 Lactate + Mito 2 pGlcNAc- 23 16 54 63Lactate + Mito 3 pGlcNAc- 72 99 Term Term Lactate + Mito 4 pGlcNAc- 2754 140 203 Lactate control 5 pGlcNAc- 30 54 96 140 Lactate control 6pGlcNAc- 30 58 200 221 Lactate control 7 Nothing 48 75 126 300 8 Nothing44 80 207 Dead 9 Propylene 49 86 180 216 glycol

[0454] % Increase in Size Day 0 Day 3 Day 5 Day 8 1 pGlcNAc- 0 0  83 135Lactate + Mito 2 pGlcNAc- 0 −30 135 174 Lactate + Mito 3 pGlcNAc- 0 38Term Term Lactate + Mito 4 pGlcNAc- 0 100 419 652 Lactate control 5pGlcNAc- 0 80 220 367 Lactate control 6 pGlcNAc- 0 93 567 637 Lactatecontrol 7 Nothing 0 56 163 525 8 Nothing 0 82 370 Dead 9 Propylene 0 76267 341 glycol

[0455] The data comparing p-GlcNAc-lactate Mito treated animals withcontrols are shown in FIGS. 29-30. The data summarized in Table XI andFIGS. 29-30 clearly suggest that the tumors rowing in the rats treatedwith the Mitomycin-containing p-GlcNAc-lactate gels animals have asignificantly retarded-rate of growth. Their growth was slowed 4-fold incomparison to the p-GlcNAc-lactate gel controls and 4-fold compared tothe no treatment controls.

20.2.3. p-GlcNAc Membrane 5′FU

[0456] Next, experiments designed to study the effect of p-GlcNAcmembrane 5′FU drug delivery systems on skin cancer tumor size wereconducted, as described, above, in Section 20.1.

[0457] The tumor volume data obtained during the study, includingpercent change in volume caused by the different treatments, issummarized in Table XII, below. Tumors were assumed to be cylindrical inshape. Their volumes were determined by measuring their width andlength, and using the following equation: V=πr²l, where the radius r is0.5 times the width and l is the length. TABLE XII p-GlcNAc Membrane +5′-Fluorouracil Animal Data Tumor Volume Tumor Volume Pre-TreatmentPost-treatment Animal # Treatment (mm³) (mm³) % Change Comments A. 5′-FUHigh Dose:  1. 5′-FU HD 393 283 −28.0  2. 5′-FU HD 785 308 −60.8  3.5′-FU HD 98.1 62.8 −36.0  4. 5′-FU HD 785 550 −30.0 Average −38.7 perAnimal B. 5′-FU Low Dose:  5. 5′-FU LD 603 170 −71.9  6. 5′-FU LD 603615 2.0  7. 5′-FU LD 269 198 −26.0  8. 5′-FU LD 169 226 33.3 Average−15.7 per animal C. p-GlcNAc Control:  9. p-GLcNAc Control 170 550 320.010. p-GLcNAc Control Died day 12 Average 320.0 per Animal D. No TreamentControl: 11. No Treatment 402 864 215.0 Sat. tumors 12. No Treatment21.2 572 2700.0 Sat. tumors Average 1457.0 per Animal E. 5′-FU viaIntravenous Injection - Control: 13. Low Dose 402 703 175.0 14. Low Dose402 402 0.0 Died day 13 15. High Dose 402 132 −67.0 Died day 13

[0458]FIG. 31 summarizes a portion of the data presented, above, inTable XII. as shown in FIG. 31, the data strongly suggest that tumorstreated with the high dose (HD) 5′-FU-containing p-GlcNAc membranes havestopped growing and have, in all cases, actually become significantlysmaller. The low dose (LD) polymer materials resulted in diseasestability and slight decrease in tumor size. In contrast, the tumors inthe control animals continued to rapidly increase in size. It isinteresting to note that two of the three control animals which weretreated via IV died during the study, indicating that systemic deliveryof the equivalent amount of 5′-FU is lethal, whereas site-specificdelivery via the p-GlcNAc polymer is efficacious in ridding the animalof the disease.

20.3. Conclusion

[0459] The data presented in this Section strongly suggest that thesite-specific delivery of anti-tumor drugs has a positive effect inretarding and reversing tumor growth. Successful results were obtainedusing p-GlcNAc drug delivery compositions having two differentformulations, namely p-GlcNAc-lactate and p-GlcNAc membraneformulations. Further, successful results were obtained using twodifferent anti-tumor drugs, 5′-FU and Mito. Thus, the p-GlcNAc drugdelivery systems of the invention exhibit anti-tumor activity, useful,for example, in the delivery of drugs specifically to the site of thetumor cells of interest.

21. EXAMPLE: FURTHER p-GlcNAc DRUG DELIVERY SYSTEMS

[0460] Described herein are further studies demonstrating the successfuluse of p-GlcNAc materials to deliver 5′-FU to the site of malignantcolon cancer tumors such that the delivered 5′-FU exhibit a therapeuticimpact upon the tumors.

21.1. Materials and Methods

[0461] p-GlcNAc Membrane 5′FU Delivery Composition:

[0462] The p-GlcNAc membrane 5′-FU delivery composition was formulatedas described herein.

[0463] The aqueous p-GlcNAc suspension used was a 1.0-1.1 mg/ml mixture.0.70 ml of the p-GlcNAc suspension was filtered using a glass frittereddisk funnel and a 0.8 micron, 47 mm membrane filter. After approximately5-6 minutes on the filtering apparatus, the membrane filter, containingthe p-GlcNAc, was removed and placed in a clean petri dish. Using aclean scalpel and forceps, the p-GlcNAc was scraped gently from themembrane and placed into a 2 ml cryovial. At this time, the appropriateamount of 5′-FU (i.e., 50 mg/ml) was added to the p-GlcNAc in thecryovial. For preparation of the control disk, no 5′-FU was added.

[0464] For example, to prepare a 0.5× disk, 0.17 ml of 5′-FU (50 mg/ml5′-FU solution) was added for a total of 8 mg of 5′-FU/disk. Thecryovial was then securely capped and the contents vortexed until athick slurry formed. The slurry was then placed into one well of a48-well plate, and flattened using a glass stirring rod. The aboveprocedure was then repeated until the desired number of p-GlcNAc diskswere prepared. After all the disks were placed in the 48-well plate, theplate was covered with Parafilm™ and the contents were frozen for atleast one hour. Then, the disks were lyophilized overnight(approximately sixteen hours). Using a sterile needle, the disks werefreed from the 48-well plate and flattened using the bottom of a cleandish and light pressure by hand. The disks were stored in clean petridishes and labeled. It should be noted that autoclaving (e.g., 20 min,121° C.) the does not damage them.

[0465] The 1× disks were prepared in the same manner as above exceptthat 0.34 ml of 5′-FU (50 mg/ml 5′-FU solution) was added for a total of16 mg of 5′-FU/disk. To prepare the 2×disks, 0.68 ml of 5′-FU (50 mg/mlof 5′-FU solution) was added for a total of 32 mg of 5′-FU/disk.

[0466] The resulting 0.5× disk contained a total of 8 mg 5′-FU each,which is equivalent to approximately half the normal total 14 day dosageof 5′-FU typically given to a cancer patient by intravenousadministration, normalized to the weight of the experimental mice basedon the typical dose of 5′-FU per kg of body weight for humans. 1×p-GlcNAc disks containing 5′-FU were prepared in the same manner, exceptthat the 1× p-GlcNAc disks contained 16 mg of 5′-FU, an amountequivalent to equal the normal total 14 day dose of 5′-FU typicallygiven to a cancer patient by intravenous administration, normalized tothe weight of the experimental mice based on the typical dose of 5′-FUper kg body weight for humans. 2× p-GlcNAc disks contained 32 mg of5′-FU, an amount equal to twice times the normal total 14 day dose of5′-FU typically given to a cancer patient by intravenous administration,normalized to the weight of the experimental mice based on the typicaldose of 5′-FU per kg body weight for humans.

[0467] Test Animals

[0468] The test animals for the present 5′-FU study on HT-29 colontumors were prepared in the same manner as described in Section 20.1.

[0469] Treatments

[0470] The mice were treated in the same manner as described in Section20.1 for the p-GlcNAc membrane 5′-FU study, except that the mice weresacrificed 10 days following the implant procedure.

21.2. Results

[0471] The tumor volume data obtained during the study included percentchange in volume caused by the different treatments, is summarized belowin Table XIII. Tumors were assumed to be cylindrical in shape. Theirvolumes were determined by measuring their width and length, and usingthe equation: V=πr²l, where the radius, r, is 0.5 times the width and lis the length. TABLE XIII Tumor Volume Tumor Volume Pre-TreatmentPost-treatment Animal # Treatment (mm³) (mm³) % Change Comments A. 0.5xDose  1. 5′-FU (.5x) 169.0 — — Died post-op  2. 5′-FU (.5x) 785.0 401.9−48  3. 5′-FU (.5x) 863.0 141.3 −83 Average −65.5 per Animal B. 1x Dose 4. 5′-FU (1.0x) 269.0 98 −63 Died Day 8 post membrane  5. 5′-FU (1.0x)269.0 — — Died Post-Op  6. 5′-FU (1.0x) 401.9 62 −84 Died Day 8 postmembrane Average −73.5 per Animal C. 2x Dose:  7. 5′-FU (2.0x) 401.998.0 −75 Died Day 8 post membrane  8. 5′-FU (2.0x) 785 50.24 −93 DiedDay 8 post membrane  9. 5′-FU (2.0x) 572 — — Died post-op Average −84.0per Animal D. p-GlcNAc Control: 10. p-GlcNAc Control 401.9 785.0 +19011. p-GlcNAc Control 1356.5 1140.0 +30 2nd mass 385.0 Average +110 perAnimal E. No Treatment Control: 12. No Treatment 401.9 1020.5 +250 13.No Treatment 50.0 572.0 +1100 Average +675 per Animal

21.3. Conclusion

[0472] As is shown in Section 21.2, above, each of the disks (i.e., 1×and 2×) were very effective in reducing tumor size. While the 0.5× dosedisks did so with little noticeable toxicity, the 1× and 2× dose disksresulted in significant mortality rates. This is presumably due to thehigher effective 5′-FU dose being delivered to the animals. Thus,according to this data, it appears that doses smaller than conventionaldoses (in this case half the conventional dose) provide site-deliveryeffective in reducing tumor size without toxic effects.

[0473] Furthermore, preparation of the disks as described in thisExample, in comparison to the Example presented in Section 20, below,appears to produce disks with less variability in dosage. Thisobservation is based on the lack of toxicity found in the data presentedin Section 20 (suggesting less incorporation of 5′-FU then originallypresumed) and the variability in tumor reduction. For example, inSection 20, Table XII shows a wide range of tumor size changes, while incontrast, no such large variations were noted in this Section, asexemplified in Table XIII, above.

22. EXAMPLE: WOUND HEALING STUDIES

[0474] The experiment described herein studies the efficacy of thep-GlcNAc materials of the invention for the promotion of wound healingand the reduction of scar tissue.

22.1. Materials and Methods

[0475] p-GlcNAc membrane: p-GlcNAc membranes (2.0×2.0 cm) were used inthis study. The p-GlcNAc was prepared using the chemical purificationmethod described, above, in Section 5.3.2.

[0476] Test Animals: Four healthy 23-27 kg, 13 week old Yorkshire pigswere used in the study. The animals were held in quarantine for sevendays prior to the surgical procedure. Each animal was identified with anindividual permanent ear tag.

[0477] Experimental Design: Four healthy Yorkshire pigs were used witheach animal serving as its own control. Under general anesthesia four2.0×2.0 cm serosal abrasion lesions were created on the surface of theintestine in each animal. Lesions 1 and 3 were left as untreated controlsites, while sites 2 and 4 were each topically treated with the testarticle. Animals were recovered for 21 days at which time they werereanesthetized. The abdomen was explored and the abrasion sites wereexamined macroscopically for hemorrhaging, necrosis, discolorations andfibroblasts. Tissue samples were collected for histomicroscopy andscanning electron microscopy. The animals were anesthetized using acombination of xylazine (3.5-5.5 mg/kg, body weight) and Telazol(5.5-8.0 mg/kg, body weight) administered intra-muscularly. Animals werethen incubated and maintained under general anesthesia using halothaneand oxygen.

[0478] Surgical Procedure: With the animal under general anesthesia andin drosal recumbency, the ventral abdomen was aseptically prepared forsurgery. The abdomen was clipped and then a gross and sterile scrub wasperformed using povidone iodine and 70% isopropyl alcohol. The animalwas then draped for surgery. A 12 cm ventral midline celiotomy was made.Four (2.0×2.0 cm) serosal abrasions were created by lightly passing anew scalpel blade (#10 Bard-Parker blade, using 1 blade/site) across theserosal surface for 200 counts. Lesions created consisted of uniformlydistributed minute, petechial hemorrhages. The first two lesions werecreated on the antimesenteric serosal surface of the small intestine andthe remaining two were created on the antimesenteric serosal surface ofthe spiral colon. The margins of each lesion site were identified byusing 4 peripherally placed sutures of 3-0 nylon. Sites 1 and 3 wereleft untreated and sites 2 and 4 were treated with the test article. Theabdomen was closed using PDS and stainless steel staples for skinclosure.

[0479] Clinical Observations were performed and recorded daily.

[0480] Euthanasia was achieved by administering an overdose ofbarbiturate while the animal was under general anesthesia on Day 21. Theprocedure was performed following the American Veterinary MedicalAssociation guidelines.

22.2. Results

[0481] The treatment with the p-GlcNAc membrane was beneficial inpromoting wound healing and reducing scar tissue formation.Macroscopically, the non-treated control sites were characterized byextensive fibrosis and discoloration. In contrast, the p-GlcNAc treatedsites developed healthy tissue and very limited fibrosis. Representativefindings are illustrated in FIGS. 32A and 32B. (The spots are theremains of the suture material that was used to identify the margins ofthe lesions.) Histomicroscopy and scanning electron microscopy findingswere consistent with the macroscopic observations.

23. EXAMPLE: TAXOL FORMULATION

[0482] Presented in this Example is a method of preparing variousconcentrations of taxol-pGlcNAc formulations.

23.1. Materials and Methods

[0483] p-GlcNAc starting material: An aqueous p-GlcNAc suspension(1.0-1.1 mg/ml mixture) was used. 0.70 ml of the aqueous p-GlcNAcsolution was filtered using a glass filtered disk funnel and a 0.8micron, 47 mm membrane filter. After approximately 5-6 minutes on thefiltering apparatus, the membrane filter, containing the p-GlcNAc, wasremoved and placed in a clean petri dish. Using a clean scalpel andforceps, the p-GlcNAc was scraped gently from the membrane and placedinto a 2 ml cryovial. At this time, the appropriate amount of taxol (6mg/ml) and H₂O (deionized and distilled) was added to the p-GlcNAc inthe cryovial.

[0484] For example, as shown in Table XIV, below, to prepare a 1× diskcontaining 0.21 mg of taxol, 35 μl of taxol (6 mg/ml taxol solution) and105 μl of H₂O were added. The cryovial was then securely capped and thecontents vortexed until a thick slurry formed. The slurry was thenplaced into a well of a 48-well plate, and flattened using a glassstirring rod. The above procedure was then repeated until the desirednumber of p-GlcNAc disks were prepared.

[0485] After all the disks were placed in the 48-well plate, the platewas covered with Parafilm™ and the contents were frozen for at least onehour. Then, the disks were lyophilized overnight (approximately sixteenhours). Using a sterile needle, the disks were freed from the 48-wellplate and flattened using the bottom of a clean dish and light pressureby hand. Disks were stored in clean petri dishes and labeled. It shouldbe noted that autoclaving (e.g., 20 min, 121° C.) the disks does notharm them.

[0486] Similarly, a 2× formulation containing 0.42 mg of taxol, wasprepared as above, except that 70 μl of taxol (mg/ml taxol solution) and105 μl H₂O was added to the p-GlcNAc in the cryovial. Table XIV, below,summarizes various taxol/p-GlcNAc formulations. TABLE XIV Dose of Taxolp-GlcNAc H₂O added Controls - No Taxol 7 ml p-GlcNAc 140 μl H₂O 1X - 35μl (0.21 mg) Taxol 7 ml p-GlcNAc 105 μl H₂O 2X - 70 μl (0.42 mg) Taxol 7ml p-GlCNAc  70 μl H₂O 4X - 140 μl (0.84 mg) Taxol 7 ml p-GlcNAc  0 μlH₂O

[0487] It is apparent that many modifications and variations of thisinvention as set forth here may be made without departing from thespirit and scope thereof. The specific embodiments described above aregiven by way of example only, and the invention is limited only by theterms of the appended claims.

What is claimed is:
 1. A method for immunoisolation of a cellcomprising: coating a cell/poly-β-1→4-N-acetylglucosamine species, saidcell/poly-β-1→4-N-acetylglucosamine species comprising apoly-β-1→4-N-acetylglucosamine species comprising about 4,000 to about150,000 N-acetylglucosamine monosaccharides covalently attached in aβ-1→4 conformation, free of protein, substantially free of other organiccontaminants, substantially free of inorganic contaminants, and having amolecular weight of about 800,000 daltons to about 30 million daltonswithin with at least one cell is encapsulated, with a coating having apolyelectrolyte charge opposite to the charge of thecell/poly-β-1→4-N-acetylglucosamine species, so that the cell within thecell/poly-β-1→4-N-acetylglucosamine species is immunoisolated.
 2. Themethod of claim 1, wherein at least one acetylglucosamine monosaccharideof the poly-β-1→4-N-acetylglucosamine species as been deacetylated.